Hyperbaric oxygen promotes bone regeneration by activating the mechanosensitive Piezo1 pathway in osteogenic progenitors.

Background: Hyperbaric oxygen (HBO) therapy is widely used to treat bone defects, but the correlation of high oxygen concentration and pressure to osteogenesis is unclear. Methods: Bilateral monocortical tibial defect surgeries were performed on 12-week-old Prrx1-Cre; Rosa26-tdTomato and Prrx1-Cre; Piezo1fl/+ mice. Daily HBO treatment was applied on post-surgery day (PSD) 1-9; and daily mechanical loading on tibia was from PSD 5 to 8. The mice were euthanized on PSD 10, and bone defect repair in their tibias was evaluated using µCT, biomechanical testing, and immunofluorescence deep-tissue imaging. The degree of angiogenesis-osteogenesis coupling was determined through spatial correlation analysis. Bone marrow stromal cells from knockout mice were cultured in vitro, and their osteogenic capacities of the cells were assessed. The activation of genes in the Piezo1-YAP pathway was evaluated using RNA sequencing and quantitative real-time polymerase chain reaction. Results: Lineage tracing showed HBO therapy considerably altered the number of Prrx1+ cells and their progeny in a healing bone defect. Using conditional knockdown mice, we found that HBO stimulation activates the Piezo1-YAP axis in Prrx1+ cells and promotes osteogenesis-angiogenesis coupling during bone repair. The beneficial effect of HBO was similar to that of anabolic mechanical stimulation, which also acts through the Piezo1-YAP axis. Subsequent transcriptome sequencing results revealed that similar mechanosensitive pathways are activated by HBO therapy in a bone defect. Conclusion: HBO therapy promotes bone tissue regeneration through the mechanosensitive Piezo1-YAP pathway in a population of Prrx1+ osteogenic progenitors. Our results contribute to the understanding of the mechanism by which HBO therapy treats bone defects. The Translational Potential of this Article: Hyperbaric oxygen therapy is widely used in clinical settings. Our results show that osteogenesis was induced by the activation of the Piezo1-YAP pathway in osteoprogenitors after HBO stimulation, and the underlying mechanism was elucidated. These results may help improve current HBO methods and lead to the formulation of alternative treatments that achieve the same functional outcomes.

Role of Piezo1 in Terminal Density Reversal of Red Blood Cells.

Density reversal of senescent red blood cells has been known for a long time, yet the identity of the candidate ion transporter(s) causing the senescent cells to swell is still elusive. While performing fractionation of RBCs from healthy individuals in Percoll density gradient and characterization of the separated fractions, we identified a subpopulation of cells in low-density fraction (1.02% ± 0.47) showing signs of senescence such as loss of membrane surface area associated with a reduction in band 3 protein abundance, and Phosphatidylserine (PS) exposure to the outer membrane. In addition, we found that these cells are overloaded with Na+ and Ca2+. Using a combination of blockers and activators of ion pumps and channels, we revealed reduced activity of Plasma membrane Ca2+ ATPase and an increase in Ca2+ and Na+ leaks through ion channels in senescent-like cells. Our data revealed that Ca2+ overload in these cells is a result of reduced PMCA activity and facilitated Ca2+ uptake via a hyperactive Piezo1 channel. However, we could not exclude the contribution of other Ca2+-permeable ion channels in this scenario. In addition, we found, as a universal mechanism, that an increase in intracellular Ca2+ reduced the initially high selectivity of Piezo1 channel for Ca2+ and allowed higher Na+ uptake, Na+ accumulation, and swelling.

Piezo ion channels: long-sought-after mechanosensors mediating hypertension and hypertensive nephropathy.

Recent advances in mechanobiology and the discovery of mechanosensitive ion channels have opened a new era of research on hypertension and related diseases. Piezo1 and Piezo2, first reported in 2010, are regarded as bona fide mechanochannels that mediate various biological and pathophysiological phenomena in multiple tissues and organs. For example, Piezo channels have pivotal roles in blood pressure control, triggering shear stress-induced nitric oxide synthesis and vasodilation, regulating baroreflex in the carotid sinus and aorta, and releasing renin from renal juxtaglomerular cells. Herein, we provide an overview of recent literature on the roles of Piezo channels in the pathogenesis of hypertension and related kidney damage, including our experimental data on the involvement of Piezo1 in podocyte injury and that of Piezo2 in renin expression and renal fibrosis in animal models of hypertensive nephropathy. The mechanosensitive ion channels Piezo1 and Piezo2 play various roles in the pathogenesis of systemic hypertension by acting on vascular endothelial cells, baroreceptors in the carotid artery and aorta, and the juxtaglomerular apparatus. Piezo channels also contribute to hypertensive nephropathy by acting on mesangial cells, podocytes, and perivascular mesenchymal cells.

PIEZO1 targeting in macrophages boosts phagocytic activity and foam cell apoptosis in atherosclerosis.

The rising incidences of atherosclerosis have necessitated efforts to identify novel targets for therapeutic interventions. In the present study, we observed increased expression of the mechanosensitive calcium channel Piezo1 transcript in mouse and human atherosclerotic plaques, correlating with infiltration of PIEZO1-expressing macrophages. In vitro administration of Yoda1, a specific agonist for PIEZO1, led to increased foam cell apoptosis and enhanced phagocytosis by macrophages. Mechanistically, PIEZO1 activation resulted in intracellular F-actin rearrangement, elevated mitochondrial ROS levels and induction of mitochondrial fragmentation upon PIEZO1 activation, as well as increased expression of anti-inflammatory genes. In vivo, ApoE-/- mice treated with Yoda1 exhibited regression of atherosclerosis, enhanced stability of advanced lesions, reduced plaque size and necrotic core, increased collagen content, and reduced expression levels of inflammatory markers. Our findings propose PIEZO1 as a novel and potential therapeutic target in atherosclerosis.

Mitigating aging and doxorubicin induced bone loss in mature mice via mechanobiology based treatments.

Aging leads to a reduced anabolic response to mechanical stimuli and a loss of bone mass and structural integrity. Chemotherapy agents such as doxorubicin exacerbate the degeneration of aging skeleton and further subject older cancer patients to a higher fracture risk. To alleviate this clinical problem, we proposed and tested a novel mechanobiology-based therapy. Building upon prior findings that i) Yoda1, the Piezo1 agonist, promoted bone growth in young adult mice and suppressed bone resorption markers in aged mice, and ii) moderate tibial loading protected bone from breast cancer-induced osteolysis, we hypothesized that combined Yoda1 and moderate loading would improve the structural integrity of adult and aged skeletons in vivo and protect bones from deterioration after chemotherapy. We first examined the effects of 4-week Yoda1 (dose 5 mg/kg, 5 times/week) and moderate tibial loading (4.5 N peak load, 4 Hz, 300 cycles for 5 days/week), individually and combined, on mature mice (∼50 weeks of age). Combined Yoda1 and loading was found to mitigate age-associated cortical and trabecular bone loss better than individual interventions. As expected, the non-treated controls experienced an average drop of cortical polar moment of inertia (Ct.pMOI) by -4.3 % over four weeks and the bone deterioration occurred in the majority (64 %) of the samples. Relative to no treatment, loading alone, Yoda1 alone, and combined Yoda1 and loading increased Ct.pMOI by +7.3 %, +9.5 %, +12.0 % and increased the % of samples with positive Ct.pMOI changes by +32 %, +26 %, and +43 %, respectively, suggesting an additive protection of aging-related bone loss for the combined therapy. We further tested if the treatment efficacy was preserved in mature mice following two weeks (six injections) of doxorubicin at the dose of 2.5 or 5 mg/kg. As expected, doxorubicin increased osteocyte apoptosis, altered bone remodeling, and impaired bone structure. However, the effects induced by DOX were too severe to be rescued by Yoda1 and loading, alone or combined, although loading and Yoda1 individually, or combined, increased the number of mice showing positive responsiveness by 0 %, +15 %, and +29 % relative to no intervention after doxorubicin exposure. Overall, this study supported the potentials and challenges of the Yoda1-based strategy in mitigating the detrimental skeletal effects caused by aging and doxorubicin.

Mechanosensitive Piezo1 channels promote neurogenic bladder fibrosis via regulating TGF-ß1/smad and Hippo/YAP1 pathways.

Bladder fibrosis is the final common pathway of neurogenic bladder (NB), and its underlying mechanisms are not fully understood. The current study aims to evaluate the involvement of Piezo1, a mechanosensitive channel, in bladder fibrosis. A full-thickness bladder specimen was taken during ileocystoplasty or ureteral reimplantation from the surgical cut's edge. By chopping off the bilateral lumbar 6 (L6) and sacral 1 (S1) spinal nerves, NB rat models were produced. Utilizing both pharmacological inhibition and Piezo1 deletion, the function of Piezo1 in the TGF-ß1-induced fibrosis model of SV-HUC-1 cells was delineated. RNA-seq, immunofluorescence, immunohistochemistry (IHC), and Western blotting were used to evaluate the degrees of fibrosis and biochemical signaling pathways. Piezo1 protein expression was noticeably elevated in the human NB bladder. The abundance of Piezo1 protein in bladder of NB rats was significantly increased. RNA-seq analysis revealed that the ECM-receptor interaction signaling pathway and collagen-containing ECM were increased in spinal cord injury (SCI)-induced bladder fibrosis. Moreover, the bladder of the NB rat model showed activation of YAP1 and TGF-ß1/Smad. In SV-HUC-1 cells, siRNA suppression of Piezo1 led to profibrotic responses and activation of the TGF-ß1/Smad pathway. However, Yoda1, a Piezo1-specific agonist, significantly reduced these effects. TGF-ß1 increased Piezo1 activation and profibrotic responses in SV-HUC-1 cells. In the TGF-ß1-induced fibrosis model of SV-HUC-1 cells, the TGF-ß1/Smad pathway was activated, whereas the Hippo/YAP1 signal pathway was blocked. Inhibition of Piezo1 further prevented this process. Piezo1 is involved in the progression of NB bladder fibrosis and profibrotic alterations in SV-HUC-1 cells, likely through regulating the TGF-ß1/Smad and Hippo/YAP1 pathways.

The role of mechanosensitive ion channel Piezo1 in knee osteoarthritis inflammation.

Mechanosensitive ion channel Piezo1 is known to mediate a variety of inflammatory pathways and is also involved in the occurrence and development of many orthopedic diseases. Although its role in the inflammatory mechanism of knee osteoarthritis (KOA) has been reported, a systematic explanation is yet to be seen. This article aims to summarize the role of inflammatory responses in the pathogenesis of KOA and elucidate the mechanism by which the Piezo1-mediated inflammatory response contributes to the pathogenesis of KOA, providing a theoretical basis for the prevention and treatment of knee osteoarthritis. The results indicate that in the mechanism leading to knee osteoarthritis, Piezo1 can mediate the inflammatory response through chondrocytes and synovial cells, participating in the pathological progression of KOA. Piezo1 has the potential to become a new target for the prevention and treatment of this disease. Additionally, as pain is one of the most severe manifestations in KOA patients, the inflammatory response mediated by Piezo1, which causes the release of inflammatory mediators and pro-inflammatory factors leading to pain, can be further explored.

Cyclic tensile stress promotes osteogenic differentiation via upregulation of Piezo1 in human dental follicle stem cells.

As periodontal progenitor cells, human dental follicle stem cells (hDFCs) play an important role in regenerative medicine research. Mechanical stimuli exert different regulatory effects on various functions of stem cells. Mechanosensitive ion channels can perceive and transmit mechanical signals. Piezo1 is a novel mechanosensitive cation channel dominated by Ca2+ permeation. The yes-associated protein 1 (YAP1) and mitogen-activated protein kinase (MAPK) pathways can respond to mechanical stimuli and play important roles in cell growth, differentiation, apoptosis, and cell cycle regulation. In this study, we demonstrated that Piezo1 was able to transduce cyclic tension stress (CTS) and promote the osteogenic differentiation of hDFCs by applying CTS of 2000 µstrain to hDFCs. Further investigation of this mechanism revealed that CTS activated Piezo1 in hDFCs and resulted in increased levels of intracellular Ca2+, YAP1 nuclear translocation, and phosphorylated protein expression levels of extracellular signalling-associated kinase 1/2 (ERK 1/2) and Jun amino-terminal kinase 1/2/3 (JNK 1/3) of the MAPK pathway family. However, when Piezo1 was knocked down in the hDFCs, all these increases disappeared. We conclude that CTS activates Piezo1 expression and promotes its osteogenesis via Ca2+/YAP1/MAPK in hDFCs. Appropriate mechanical stimulation promotes the osteogenic differentiation of hDFCs via Piezo1. Targeting Piezo1 may be an effective strategy to regulate the osteogenic differentiation of hDFCs, contributing to MSC-based therapies in the field of bone tissue engineering.

Nano-mechanical Immunoengineering: Nanoparticle Elasticity Reprograms Tumor-Associated Macrophages via Piezo1.

The mechanical properties of nanoparticles play a crucial role in regulating nanobiointeractions, influencing processes such as blood circulation, tumor accumulation/penetration, and internalization into cancer cells. Consequently, they have a significant impact on drug delivery and therapeutic efficacy. However, it remains unclear whether and how macrophages alter their biological function in response to nanoparticle elasticity. Here, we report on the nano-mechanical biological effects resulting from the interactions between elastic silica nanoparticles (SNs) and macrophages. The SNs with variational elasticity Young's moduli ranging from 81 to 837 MPa were synthesized, and it was demonstrated that M2 [tumor-associated macrophages (TAMs)] could be repolarized to M1 by the soft SNs. Additionally, our findings revealed that cell endocytosis, membrane tension, the curvature protein Baiap2, and the cytoskeleton were all influenced by the elasticity of SNs. Moreover, the mechanically sensitive protein Piezo1 on the cell membrane was activated, leading to calcium ion influx, activation of the NF-κB pathway, and the initiation of an inflammatory response. In vivo experiments demonstrated that the softest 81 MPa SNs enhanced tumor penetration and accumulation and repolarized TAMs in intratumoral hypoxic regions, ultimately resulting in a significant inhibition of tumor growth. Taken together, this study has established a cellular feedback mechanism in response to nanoparticle elasticity, which induces plasma membrane deformation and subsequent activation of mechanosensitive signals. This provides a distinctive "nano-mechanical immunoengineering" strategy for reprogramming TAMs to enhance cancer immunotherapy.

Research progress on the immunological functions of Piezo1 a receptor molecule that responds to mechanical force.

The human immune system is capable of defending against, monitoring, and self-stabilizing various immune cells. Differentiation, proliferation, and development of these cells are regulated by biochemical signals. Moreover, biophysical signals, such as mechanical forces, have been found to affect immune cell function, thus introducing a new area of immunological research. Piezo1, a mechanically sensitive ion channel, was awarded the Nobel Prize for Physiology and Medicine in 2021. This channel is present on the surface of many cells, and when stimulated by mechanical force, it controls calcium (Ca2+) inside the cells, leading to changes in downstream signals and thus regulating cell functions. Piezo1 is also expressed in various innate and adaptive immune cells and plays a major role in the immune function. In this review, we will explore the physiological functions and regulatory mechanisms of Piezo1 and its impact on innate and adaptive immunity. This may offer new insights into diagnostics and therapeutics for the prevention and treatment of diseases and surgical infections.

The mechanosensitive Piezo1 channel exacerbates myocardial ischaemia/reperfusion injury by activating caspase-8-mediated PANoptosis.

PANoptosis is a newly discovered type of cell death characterized by pyroptosis, apoptosis and/or necroptosis and has been implicated in the inflammatory response. Piezo1 is a mechanosensitive ion channel that plays important roles in physiological development and various diseases. However, whether cardiomyocytes undergo PANoptosis during myocardial ischaemia/reperfusion (I/R) injury and the role of Piezo1 in this process remain largely unexplored. In this study, our results revealed that the expression levels of the main components of the PANoptosome, including caspase-8, caspase-3, NLRP3, caspase-1, GSDMD, RIPK1, RIPK3 and MLKL, were significantly upregulated in I/R heart tissues over time, indicating the occurrence of PANoptosis in I/R hearts. Accordingly, Piezo1 expression was significantly upregulated in I/R-injured hearts and hypoxia/reoxygenation (H/R)-treated cardiomyocytes. In contrast, pharmacological inhibition of Piezo1 by the inhibitor GsMTx4 in mice markedly attenuated the I/R-mediated decline in cardiac contractile function and increases in infarct size, apoptosis, oxidative stress and inflammation accompanied by the inhibition of PANoptosis-related mediators in I/R hearts. Consistently, the effects of Piezo1 on calcium influx and PANoptosis were further verified by GsMTx4 and Piezo1 activator Yoda1 in H/R-treated cardiomyocytes in vitro. Moreover, caspase-8 rather than calcium influx was required for H/R-induced PANoptosis in vitro. Mechanistically, Piezo1 interacts with caspase-8, a key initial activator of the PANoptosome complex, which subsequently activates cardiomyocyte PANoptosis, leading to cardiac dysfunction. In summary, these data suggest that Piezo1 is a new cardiac mechanosensor that promotes cardiac I/R injury possibly through the caspase-8-mediated activation of cardiomyocyte PANoptosis and highlight that Piezo1 may represent a new target for treating ischaemic heart disease.

Effects of Panax notoginseng saponins on alleviating low shear induced endothelial inflammation and thrombosis via Piezo1 signalling.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax notoginseng saponins (PNS) are the major effective components of Panax notoginseng (burk) F.H.Chen which is one of the classic promoting blood circulation herbs in traditional Chinese medicine. PNS is widely used in China for the treatment of cerebral ischemic stroke. Pathological low shear stress is a causal factor in endothelial inflammation and thrombosis. However, the mechanism of PNS against low shear related endothelial inflammation is still unclear. AIM TO THE STUDY: This study aims to investigate the effects of PNS against endothelial inflammation induced by low shear stress and to explore the underlying mechanical and biological mechanisms. MATERIALS AND METHODS: Mouse model of carotid partial ligation for inducing low endothelial shear stress was established, the pharmacodynamic effect and mechanism of PNS against endothelial inflammation induced by low shear stress through Piezo1 were explored. Yoda1-evoked Piezo1 activation and expression in human umbilical vein endothelial cells (HUVECs) were determined at static condition. Microfluidic channel systems were used to apply shear stress on HUVECs and Piezo1 siRNA HUVECs to determine PECAM-1, p-YAP and VCAM-1 expression. And platelet rich plasma (PRP) was introduced to low shear treated endothelial cells surface to observe the adhesion and activation by fluorescence imaging and flowcytometry. RESULTS: PNS attenuated endothelial inflammation and improved blood flow in a reasonable dose response pattern in carotid partial ligation mouse model by influencing Piezo1 and PECAM-1 expression, while suppressing yes-associated protein (YAP) nuclear translocation. We found Piezo1 sensed abnormal shear stress and transduced these mechanical signals by different pathways in HUVECs, and PNS relieved endothelial inflammation induced by low shear stress through Piezo1. We also found Piezo1 signalling has interaction with PECAM-1 under low shear stress, which were involved in platelets adhesion to endothelial cells. Low shear stress increased YAP nuclear translocation and increased VCAM-1 expression in HUVECs which might activate platelets. PNS inhibited low shear induced Piezo1 and PECAM-1 expression and YAP nuclear translocation in HUVECs, furthermore inhibited platelet adhesion and activation on dysfunctional endothelial cells induced by low shear stress. CONCLUSION: PNS ameliorated endothelial inflammation and thrombosis induced by low shear stress through modulation of the Piezo1 channel, PECAM-1 expression, and YAP nuclear translocation. PNS might serve as a potential therapeutic candidate for ameliorating endothelial inflammation induced by abnormal blood shear stress.

Piezo1 Activation Drives Enhanced Collagen Synthesis in Aged Animal Skin Induced by Poly L-Lactic Acid Fillers.

Poly L-lactic acid (PLLA) fillers stimulate collagen synthesis by activating various immune cells and fibroblasts. Piezo1, an ion channel, responds to mechanical stimuli, including changes in extracellular matrix stiffness, by mediating Ca2+ influx. Given that elevated intracellular Ca2+ levels trigger signaling pathways associated with fibroblast proliferation, Piezo1 is a pivotal regulator of collagen synthesis and tissue fibrosis. The aim of the present study was to investigate the impact of PLLA on dermal collagen synthesis by activating Piezo1 in both an H2O2-induced cellular senescence model in vitro and aged animal skin in vivo. PLLA elevated intracellular Ca2+ levels in senescent fibroblasts, which was attenuated by the Piezo1 inhibitor GsMTx4. Furthermore, PLLA treatment increased the expression of phosphorylated ERK1/2 to total ERK1/2 (pERK1/2/ERK1/2) and phosphorylated AKT to total AKT (pAKT/AKT), indicating enhanced pathway activation. This was accompanied by upregulation of cell cycle-regulating proteins (CDK4 and cyclin D1), promoting the proliferation of senescent fibroblasts. Additionally, PLLA promoted the expression of phosphorylated mTOR/S6K1/4EBP1, TGF-ß, and Collagen I/III in senescent fibroblasts, with GsMTx4 treatment mitigating these effects. In aged skin, PLLA treatment similarly upregulated the expression of pERK1/2/ERK1/2, pAKT/AKT, CDK4, cyclin D1, mTOR/S6K1/4EBP1, TGF-ß, and Collagen I/III. In summary, our findings suggest Piezo1's involvement in PLLA-induced collagen synthesis, mediated by heightened activation of cell proliferation signaling pathways such as pERK1/2/ERK1/2, pAKT/AKT, and phosphorylated mTOR/S6K1/4EBP1, underscoring the therapeutic potential of PLLA in tissue regeneration.

Radial extracorporeal shockwave promotes osteogenesis-angiogenesis coupling of bone marrow stromal cells from senile osteoporosis via activating the Piezo1/CaMKII/CREB axis.

Radial extracorporeal shockwave (r-ESW) and bone marrow stromal cells (BMSCs) have been reported to alleviate senile osteoporosis (SOP), but its regulatory mechanism remains unclear. In this study, we firstly isolated human BMSCs from bone marrow samples and treated with varying r-ESW doses. And we found that r-ESW could enhance the proliferation of SOP-BMSCs in a dose-dependent manner by EdU assay. Subsequently, the impact of r-ESW on the proliferation, apoptosis and multipotency of BMSCs was assessed. And the outcomes of flow cytometry, Alizarin red S (ARS), and tube formation test demonstrated that the optimal shockwave obviously boosted SOP-BMSCs osteogenesis and angiogenesis but exhibited no significant impact on cell apoptosis. Additionally, the signaling of Piezo1 and CaMKII/CREB was examined by Western blotting, qPCR and immunofluorescence. And the results showed that r-ESW promoted the expression of Piezo1, increased intracellular Ca2+ and activated the CaMKII/CREB signaling pathway. Then, the application of Piezo1 siRNA hindered the r-ESW-induced enhancement ability of osteogenesis coupling with angiogenesis of SOP-BMSCs. The use of the CaMKII/CREB signaling pathway inhibitor KN93 suppressed the Piezo1-induced increase in osteogenesis and angiogenesis in SOP-BMSCs. Finally, we also found that r-ESW might alleviate SOP in the senescence-accelerated mouse prone 6 (SAMP6) model by activating Piezo1. In conclusion, our research offers experimental evidence and an elucidated underlying molecular mechanism to support the use of r-ESW as a credible rehabilitative treatment for senile osteoporosis.

PIEZO1 activation may serve as an early tissue biomarker for the prediction of irradiation-induced salivary gland dysfunction.

Irradiation (IR)-induced xerostomia is the most common side effect of radiation therapy in patients with head and neck cancer (HNC). Xerostomia diagnosis is mainly based on the patient's medical history and symptoms. Currently, no direct biomarkers are available for the early prediction of IR-induced xerostomia. Here, we identified PIEZO1 as a novel predictive tissue biomarker for xerostomia. Our data demonstrate that PIEZO1 is significantly upregulated at the gene and protein levels during IR-induced salivary gland (SG) hypofunction. Notably, PIEZO1 upregulation coincided with that of inflammatory (F4/80) and fibrotic markers (fibronectin and collagen fibers accumulation). These findings suggest that PIEZO1 upregulation in SG tissue may serve as a novel predictive marker for IR-induced xerostomia.

Guizhitongluo Tablet inhibits atherosclerosis and foam cell formation through regulating Piezo1/NLRP3 mediated macrophage pyroptosis.

BACKGROUND: Atherosclerosis (AS) is the main pathological basis for the development of cardiovascular diseases. Vascular inflammation is an important factor in the formation of AS, and macrophage pyroptosis plays a key role in AS due to its unique inflammatory response. Guizhitongluo Tablet (GZTLT) has shown clinically effective in treating patients with AS, but its mechanism is elusive. PURPOSE: This study was to determine the effects of GZTLT on atherosclerotic vascular inflammation and pyroptosis and to understand its underlying mechanism. MATERIALS AND METHODS: The active constituents of GZTLT were analysed by means of UPLC-HRMS. In vivo experiments were performed using ApoE-/- mice fed a high fat diet for 8 weeks, followed by treatment with varying concentrations of GZTLT orally by gavage and GsMTx4 (GS) intraperitoneally and followed for another 8 weeks. Oil red O, Haematoxylin-eosin (HE) and Masson staining were employed to examine the lipid content, plaque size, and collagen fibre content of the mouse aorta. Immunofluorescence staining was utilised to identify macrophage infiltration, as well as the expression of Piezo1 and NLRP3 proteins in aortic plaques. The levels of aortic inflammatory factors were determined using RT-PCR and ELISA. In vitro, foam cell formation in bone marrow-derived macrophages (BMDMs) was observed using Oil Red O staining. Intracellular Ca2+ measurements were performed to detect the calcium influx in BMDMs, and the expression of NLRP3 and its related proteins were detected by Western blot. RESULTS: The UPLC-HRMS analysis revealed 31 major components of GZTLT. Our data showed that GZTLT inhibited aortic plaque formation in mice and increased plaque collagen fibre content to stabilise plaques. In addition, GZTLT could restrain the expression of serum lipid levels and suppress macrophage foam cell formation. Further studies found that GZTLT inhibited macrophage infiltration in aortic plaques and suppressed the expression of inflammatory factors. It is noteworthy that GZTLT can restrain Piezo1 expression and reduce Ca2+ influx in BMDMs. Additionally, we found that GZTLT could regulate NLRP3 activation and pyroptosis by inhibiting Piezo1. CONCLUSION: The present study suggests that GZTLT inhibits vascular inflammation and macrophage pyroptosis through the Piezo1/NLRP3 signaling pathway, thereby delaying AS development. Our finding provides a potential target for AS treatment and drug discovery.

Fluid shear stress-mediated Piezo1 alleviates osteocyte apoptosis by activating the PI3K/Akt pathway.

Glucocorticoid-induced osteoporosis serves as a primary cause for secondary osteoporosis and fragility fractures, representing the most prevalent adverse reaction associated with prolonged glucocorticoid use. In this study, to elucidate the impact and underlying mechanisms of fluid shear stress (FSS)-mediated Piezo1 on dexamethasone (Dex)-induced apoptosis, we respectively applied Dex treatment for 6 h, FSS at 9 dyne/cm2 for 30 min, Yoda1 treatment for 2 h, and Piezo1 siRNA transfection to intervene in MLO-Y4 osteocytes. Western blot analysis was used to assess the expression of Cleaved Caspase-3, Bax, Bcl-2, and proteins associated with the PI3K/Akt pathway. Additionally, qRT-PCR was utilized to quantify the mRNA expression levels of these molecules. Hoechst 33258 staining and flow cytometry were utilized to evaluate the apoptosis levels. The results indicate that FSS at 9 dyne/cm2 for 30 min significantly upregulates Piezo1 in osteocytes. Following Dex-induced apoptosis, the phosphorylation levels of PI3K and Akt are markedly suppressed. FSS-mediated Piezo1 exerts a protective effect against Dex-induced apoptosis by activating the PI3K/Akt pathway. Additionally, downregulating the expression of Piezo1 in osteocytes using siRNA exacerbates Dex-induced apoptosis. To further demonstrate the role of the PI3K/Akt signaling pathway, after intervention with the PI3K pathway inhibitor, the activation of the PI3K/Akt pathway by FSS-mediated Piezo1 in osteocytes was significantly inhibited, reversing the anti-apoptotic effect. This study indicates that under FSS, Piezo1 in MLO-Y4 osteocytes is significantly upregulated, providing protection against Dex-induced apoptosis through the activation of the PI3K/Akt pathway.

Quercetin ameliorates atherosclerosis by inhibiting inflammation of vascular endothelial cells via Piezo1 channels.

BACKGROUND: Natural antioxidants, exemplified by quercetin (Qu), have been shown to exert a protective effect against atherosclerosis (AS). However, the precise pharmacological mechanisms of Qu also remain elusive. PURPOSE: Here, we aimed to uncover the anti-atherosclerotic mechanisms of Qu. METHODS/STUDY DESIGNS: The inflammatory cytokine expression, activity of NLRP3 inflammasome and NF-κB, as well as mechanically activated currents and intracellular calcium levels were measured in endothelial cells (ECs). In addition, to explore whether Qu inhibited atherosclerotic plaque formation via Piezo1 channels, Ldlr-/- and Piezo1 endothelial-specific knockout mice (Piezo1△EC) were established. RESULTS: Our findings revealed that Qu significantly inhibited Yoda1-evoked calcium response in human umbilical vein endothelial cells (HUVECs), underscoring its role as a selective modulator of Piezo1 channels. Additionally, Qu effectively reduced mechanically activated currents in HUVECs. Moreover, Qu exhibited a substantial inhibitory effect on inflammatory cytokine expression and reduced the activity of NF-κB/NLRP3 in ECs exposed to ox-LDL or mechanical stretch, and these effects remained unaffected after Piezo1 genetic depletion. Furthermore, our study demonstrated that Qu substantially reduced the formation of atherosclerotic plaques, and this effect remained consistent even after Piezo1 genetic depletion. CONCLUSION: These results collectively provide compelling evidence that Qu ameliorates atherosclerosis by inhibiting the inflammatory response in ECs by targeting Piezo1 channels. In addition, Qu modulated atherosclerosis via inhibiting Piezo1 mediated NFκB/IL-1ß and NLRP3/caspase1/ IL-1ß axis to suppress the inflammation. Overall, this study reveals the potential mechanisms by which natural antioxidants, such as Qu, protect against atherosclerosis.

The mechanosensitive ion channel Piezo1 contributes to podocyte cytoskeleton remodeling and development of proteinuria in lupus nephritis.

Piezo1 functions as a special transducer of mechanostress into electrochemical signals and is implicated in the pathogenesis of various diseases across different disciplines. However, whether Piezo1 contributes to the pathogenesis of lupus nephritis (LN) remains elusive. To study this, we applied an agonist and antagonist of Piezo1 to treat lupus-prone MRL/lpr mice. Additionally, a podocyte-specific Piezo1 knockout mouse model was also generated to substantiate the role of Piezo1 in podocyte injury induced by pristane, a murine model of LN. A marked upregulation of Piezo1 was found in podocytes in both human and murine LN. The Piezo1 antagonist, GsMTx4, significantly alleviated glomerulonephritis and tubulointerstitial damage, improved kidney function, decreased proteinuria, and mitigated podocyte foot process effacement in MRL/lpr mice. Moreover, podocyte-specific Piezo1 deletion showed protective effects on the progression of proteinuria and podocyte foot process effacement in the murine LN model. Mechanistically, Piezo1 expression was upregulated by inflammatory cytokines (IL-6, TNF-α and IFN-γ), soluble urokinase Plasminogen Activator Receptor and its own activation. Activation of Piezo1 elicited calcium influx, which subsequently enhanced Rac1 activity and increased active paxillin, thereby promoting cytoskeleton remodeling and decreasing podocyte motility. Thus, our work demonstrated that Piezo1 contributed to podocyte injury and proteinuria progression in LN. Hence, targeted therapy aimed at decreasing or inhibiting Piezo1 could represent a novel strategy to treat LN.

Piezo1 ion channels are capable of conformational signaling.

Piezo1 is a mechanically activated ion channel that senses forces with short latency and high sensitivity. Piezos undergo large conformational changes, induce far-reaching deformation onto the membrane, and modulate the function of two-pore potassium (K2P) channels. Taken together, this led us to hypothesize that Piezos may be able to signal their conformational state to other nearby proteins. Here, we use chemical control to acutely restrict Piezo1 conformational flexibility and show that Piezo1 conformational changes, but not ion permeation through them, are required for modulating the K2P channel K2P2.1 (TREK1). Super-resolution imaging and stochastic simulations further reveal that both channels do not co-localize, which implies that modulation is not mediated through direct binding interactions; however, at high Piezo1 densities, most TREK1 channels are within the predicted Piezo1 membrane footprint, suggesting that the footprint may underlie conformational signaling. We speculate that physiological roles originally attributed to Piezo1 ionotropic function could, alternatively, involve conformational signaling.