Ion Channel Piezo1 Research Articles

PIEZO Ion Channels in Cardiovascular Functions and Diseases.

The cardiovascular system provides blood supply throughout the body and as such is perpetually applying mechanical forces to cells and tissues. Thus, this system is primed with mechanosensory structures that respond and adapt to changes in mechanical stimuli. Since their discovery in 2010, PIEZO ion channels have dominated the field of mechanobiology. These have been proposed as the long-sought-after mechanosensitive excitatory channels involved in touch and proprioception in mammals. However, more and more pieces of evidence point to the importance of PIEZO channels in cardiovascular activities and disease development. PIEZO channel-related cardiac functions include transducing hemodynamic forces in endothelial and vascular cells, red blood cell homeostasis, platelet aggregation, and arterial blood pressure regulation, among others. PIEZO channels contribute to pathological conditions including cardiac hypertrophy and pulmonary hypertension and congenital syndromes such as generalized lymphatic dysplasia and xerocytosis. In this review, we highlight recent advances in understanding the role of PIEZO channels in cardiovascular functions and diseases. Achievements in this quickly expanding field should open a new road for efficient control of PIEZO-related diseases in cardiovascular functions.

Simulations of Functional Motions of Super Large Biomolecules with a Mixed-Resolution Model.

Many large protein machines function through an interplay between large-scale movements and intricate conformational changes. Understanding functional motions of these proteins through simulations becomes challenging for both all-atom and coarse-grained (CG) modeling techniques because neither approach alone can readily capture the full details of these motions. In this study, we develop a multiscale model by employing the popular MARTINI CG model to represent a heterogeneous environment and structurally stable proteins and using the united-atom (UA) model PACE to describe proteins undergoing subtle conformational changes. PACE was previously developed to be compatible with the MARTINI solvent and membrane. Here, we couple the protein descriptions of the two models by directly mixing UA and CG interaction parameters to greatly simplify parameter determination. Through extensive validations with diverse protein systems in solution or membrane, we demonstrate that only additional parameter rescaling is needed to enable the resulting model to recover the stability of native structures of proteins under mixed representation. Moreover, we identify the optimal scaling factors that can be applied to various protein systems, rendering the model potentially transferable. To further demonstrate its applicability for realistic systems, we apply the model to a mechanosensitive ion channel Piezo1 that has peripheral arms for sensing membrane tension and a central pore for ion conductance. The model can reproduce the coupling between Piezo1's large-scale arm movement and subtle pore opening in response to membrane stress while consuming much less computational costs than all-atom models. Therefore, our model shows promise for studying functional motions of large protein machines.

The role of mechanically sensitive ion channel Piezo1 in bone remodeling.

Piezo1 (2010) was identified as a mechanically activated cation channel capable of sensing various physical forces, such as tension, osmotic pressure, and shear force. Piezo1 mediates mechanosensory transduction in different organs and tissues, including its role in maintaining bone homeostasis. This review aimed to summarize the function and possible mechanism of Piezo1 in the mechanical receptor cells in bone tissue. We found that it is a potential therapeutic target for the treatment of bone diseases.

Increased PIEZO1 Expression Is Associated with Worse Clinical Outcomes in Hormone-Receptor-Negative Breast Cancer Patients.

PIEZO1 plays a crucial role in the human body as a mechanosensory ion channel. It has been demonstrated that PIEZO1 is important in tissue development and regulating many essential physiological processes. Studies have suggested that the PIEZO1 ion channel plays a role in invasion and progression in cancer; elevated levels of PIEZO1 have been correlated with increased migration in breast cancer cells, chemo-resistance and invasion in gastric cancer cells, and increased invasion of osteosarcoma cells. In addition, high PIEZO1 expression levels were correlated with a worse prognosis in glioma patients. On the other hand, studies in lung cancer have attributed high PIEZO1 levels to better patient outcomes. However, the clinical impact of PIEZO1 in breast cancer is not well characterized. Therefore, our goal was to determine the clinical relevance of PIEZO1 in breast cancer. An analysis of breast cancer data from The Cancer Genome Atlas (TCGA) was conducted to investigate PIEZO1 expression levels and correlation to survival, followed by validation in an independent dataset, GSE3494. We also performed gene set enrichment analysis (GSEA) and pathway enrichment analysis. We also analyzed the immune cell composition in breast tumors from TCGA through a CIBERSORT algorithm. Our results demonstrated that the PIEZO1 expression levels are higher in hormone-receptor (HR)-negative than in HR-positive cohorts. High PIEZO1 expression is correlated with a significant decrease in survival in HR-negative cohorts, especially in triple-negative breast cancer (TNBC), suggesting that PIEZO1 could be utilized as a prognostic biomarker in HR-negative breast cancer. GSEA showed that various signaling pathways associated with more invasive phenotypes and resistance to treatments, including epithelial-mesenchymal transition (EMT), hypoxia, and multiple signaling pathways, are enriched in high-PIEZO1 HR-negative tumors. Our results also demonstrated a decrease in CD8+ and CD4+ T cell infiltration in high-PIEZO1 HR-negative tumors. Further investigations are necessary to elucidate the mechanistic roles of PIEZO1 in HR-negative breast cancer.

Mechanical gated ion channel Piezo1: Function, and role in macrophage inflammatory response.

Macrophages are present in many mechanically active tissues and are often subjected to varying degrees of mechanical stimulation. Macrophages play a crucial role in resisting pathogen invasion and maintaining tissue homeostasis. Piezo-type mechanosensitive channel component 1 (Piezo1) is the main cation channel involved in the rapid response to mechanical stimuli in mammals. This channel plays a crucial role in controlling blood pressure and motor performance and regulates urinary osmotic pressure and epithelial cell proliferation and division. In recent years, numerous studies have shown that in macrophages, Piezo1 not only plays a role in regulating the aforementioned physiological processes but also participates in multiple pathological processes such as inflammation and cancer. In this review, we summarize the research progress on Piezo1-mediated regulation of macrophage-mediated inflammatory responses through downstream signalling pathways and the aerobic glycolysis pathway.

Chronic Ankle Joint Instability Induces Ankle Sensorimotor Dysfunction: A Controlled Laboratory Study.

Chronic ankle instability (CAI) is a clinical sequela that causes the recurrence of ankle sprain by inducing ankle sensorimotor dysfunction. Animal models of CAI have recently shown that ankle ligament injuries mimicking an ankle sprain result in chronic loss of ankle sensorimotor function. However, the underlying mechanisms determining the pathogenesis of CAI remain unclear. Ankle instability after an ankle sprain leads to the degeneration of the mechanoreceptors, resulting in ankle sensorimotor dysfunction and the development of CAI. Controlled laboratory study. Four-week-old male Wistar rats (N = 30) were divided into 2 groups: (1) the ankle joint instability (AJI) group with ankle instability induced by transecting the calcaneofibular ligament (n = 15) and (2) the sham group (n = 15). Ankle instability was assessed using the anterior drawer test and the talar tilt test at 4, 6, and 8 weeks after the operation (n = 5, for each group at each time point), and ankle sensorimotor function was assessed using behavioral tests, including ladder walking and balance beam tests, every 2 weeks during the postoperative period. Morphology and number of mechanoreceptors in the intact anterior talofibular ligament (ATFL) were histologically analyzed by immunofluorescence staining targeting the neurofilament medium chain and S100 proteins at 4, 6, and 8 weeks postoperatively (n = 5 per group). Sensory neurons that form mechanoreceptors were histologically analyzed using immunofluorescence staining targeting the mechanosensitive ion channel PIEZO2 at 8 weeks postoperatively (n = 5). Ankle sensorimotor function decreased over time in the AJI group, exhibiting decreased ankle instability compared with the sham group (P = .045). The number of mechanoreceptors in the ATFL was reduced (P < .001) and PIEZO2 expression in the sensory neurons decreased (P = .008) at 8 weeks postoperatively. The number of mechanoreceptors was negatively correlated with ankle sensorimotor dysfunction (P < .001). The AJI model demonstrated degeneration of the mechanoreceptors in the ATFL and decreased mechanosensitivity of the sensory neurons, which may contribute to CAI. Ankle instability causes degeneration of mechanoreceptors and decreases the mechanosensitivity of sensory neurons involved in the development of CAI. This finding emphasizes the importance of controlling ankle instability after ankle sprains to prevent recurrence and the onset of CAI.

Mechanical stiffness promotes skin fibrosis via Piezo1-Wnt2/Wnt11-CCL24 positive feedback loop

Skin fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) caused by fibrotic disorders of the skin. In recent years, ECM stiffness has emerged as a prominent mechanical cue that precedes skin fibrosis and drives its progression by promoting fibroblasts activation. However, how stiffness influences fibroblasts activation for skin fibrosis progression remains unknown. Here, we report a positive feedback loop mediated by the mechanosensitive ion channel Piezo1 and aberrant tissue mechanics in driving skin fibrosis. Piezo1 is upregulated in fibrotic skin in both humans and mice. Piezo1 knockdown dermal fibroblasts lose their fibroproliferative phenotypes despite being grown on a stiffer substrate. We show that Piezo1 acts through the Wnt2/Wnt11 pathway to mechanically induce secretion of C-C motif chemokine ligand 24 (CCL24, also known as eotaxin-2), a potent cytokine associated with fibrotic disorders. Importantly, adeno-associated virus (AAV)-mediated Piezo1 knockdown ameliorated the progression of skin fibrosis and skin stiffness in mice. Overall, increased matrix stiffness promotes skin fibrosis through the inflammatory Piezo1-Wnt2/Wnt11-CCL24 pathway. In turn, a stiffer skin microenvironment increases Piezo1 expression to exacerbate skin fibrosis aggression. Therefore, targeting Piezo1 represents a strategy to break the positive feedback loop between fibroblasts mechanotransduction and aberrant tissue mechanics in skin fibrosis.

Selenium-SelK-GPX4 axis protects nucleus pulposus cells against mechanical overloading-induced ferroptosis and attenuates senescence of intervertebral disc

Intervertebral disc degeneration (IVDD) is one of the most prevalent spinal degenerative disorders and imposes places heavy medical and economic burdens on individuals and society. Mechanical overloading applied to the intervertebral disc (IVD) has been widely recognized as an important cause of IVDD. Mechanical overloading-induced chondrocyte ferroptosis was reported, but the potential association between ferroptosis and mechanical overloading remains to be illustrated in nucleus pulposus (NP) cells. In this study, we discovered that excessive mechanical loading induced ferroptosis and endoplasmic reticulum (ER) stress, which were detected by mitochondria and associated markers, by increasing the intracellular free Ca2+ level through the Piezo1 ion channel localized on the plasma membrane and ER membrane in NP cells. Besides, we proposed that intracellular free Ca2+ level elevation and the activation of ER stress are positive feedback processes that promote each other, consistent with the results that the level of ER stress in coccygeal discs of aged Piezo1-CKO mice were significantly lower than that of aged WT mice. Then, we confirmed that selenium supplementation decreased intracellular free Ca2+ level by mitigating ER stress through upregulating Selenoprotein K (SelK) expression. Besides, ferroptosis caused by the impaired production and function of Glutathione peroxidase 4 (GPX4) due to mechanical overloading-induced calcium overload could be improved by selenium supplementation through Se-GPX4 axis and Se-SelK axis in vivo and in vitro, eventually presenting the stabilization of the extracellular matrix (ECM). Our findings reveal the important role of ferroptosis in mechanical overloading-induced IVDD, and selenium supplementation promotes significance to attenuate ferroptosis and thus alleviates IVDD, which might provide insights into potential therapeutic interventions for IVDD.

Hot Topics and Emerging Trends in Mechanobiology Research

Mechanobiology focuses on a series of important physiopathological processes, such as how cells perceive different mechanomechanical stimuli, the process of intracellular mechanotransduction, and how mechanical signals determine the behavior and fate of cells. From the initial stage of embryogenesis, to developmental biology and regenerative medicine, or even through the whole life process, mechanical signaling cascades and cellular mechanical responses in mechanobiology are of great significance in biomedical research. In recent years, research in the field of mechanobiology has undergone remarkable development. Several scientific consortia around the world have been analyzing mechanobiological processes from different perspectives, aiming to gain insights into the regulatory mechanisms by which mechanical factors affect cell fate determination. In this article, we summarized and reviewed the topics that have attracted more research interests in recent years in the field of mechanobiology, for example, arterial blood vessels, stem cell, and ion channel. We also discussed the potential trends that may emerge, such as nuclear deformation, fibrous extracellular matrix, tumor mechanobiology, cellular mechanotransduction, and piezo ion channels. In addition, we put forward new ideas concerning the limitations of mechanism research and the importance of big data analysis and mining in this field, thereby providing objective support and a systematic framework for grasping the hot research topics and exploring new research directions in the field of mechanobiology.

CHONDROCYTE-SPECIFIC KNOCKOUT OF PIEZO ION CHANNELS PROTECTS AGAINST POST-TRAUMATIC OSTEOARTHRITIS

Osteoarthritis (OA) is the leading cause of pain and disability worldwide and is characterized by the degenerative changes of articular cartilage. Joint loading is required for cartilage maintenance; however, hyper-physiologic loading is a risk factor for OA. Mechanosensitive ion channels Piezo1 and Piezo2 synergistically transduce hyper-physiologic compression of chondrocytes, leading to chondrocyte death and onset of OA. This injury response is inhibited by Piezo channel loss of function, however the mechanistic role of Piezo channels in vivo is unknown. We examined the hypothesis that deletion of Piezo in chondrocytes will protect mice from joint damage and pain-related behaviors following a surgical destabilization of the medial meniscus (DMM), investigating a key mechanistic and mechanobiological role of these channels in the pathogenesis of OA.Aggrecan-Cre Piezo1 and Piezo1/2 knockout mice ((Agc)1-CREERT2;Piezo1fl/flPiezo2fl/fl) were generated and given a 5-day Tamoxifen regimen at 12-weeks of age (n=6–12/group/sex). Cre-negative mice served as controls. At 16-weeks, mice received DMM surgery on the left knee. 12-weeks following DMM prior to sacrifice, activity and hyperalgesia were measured using spontaneous running wheels and a small animal algometer. Structural changes in bone, cartilage, and synovium were characterized using microCT, histology, and Modified Mankin Score criteria.Knockout of Piezo1/2 channels was chondroprotective in both sexes following DMM surgery as demonstrated by reduced Modified Mankin Score compared to control animals. Piezo1 KO was chondroprotective in only female mice, indicating a sexually dimorphic response. Piezo1 and Piezo1/2 KO was protective against pain in male mice, while females displayed no differences compared to controls. No changes were observed in bone morphology.Chondrocyte-specific Piezo1/2 knockout protects the knee joint from structural damage, hyperalgesia and functional deficits in a surgical model of PTOA in male and female mice, illustrating the importance of Piezo channels in response to injury in vivo. Future work aims to interrogate potential sexually dimorphic responses to cartilage damage and investigating Piezo2 KO mice.

Mechanosensitive Ion Channels Piezo1 and Piezo2 Mediate Motor Responses In Vivo During Transcranial Focused Ultrasound Stimulation of the Rodent Cerebral Motor Cortex.

Transcranial focused ultrasound (tFUS) neuromodulation offers a noninvasive, safe, deep brain stimulation with high precision, presenting potential in understanding neural circuits and treating brain disorders. This in vivo study investigated the mechanism of tFUS in activating the opening of the mechanosensitive ion channels Piezo1 and Piezo2 in the mouse motor cortex to induce motor responses. Piezo1 and Piezo2 were knocked down separately in the mouse motor cortex, followed by EMG and motor cortex immunofluorescence comparisons before and after knockdown under tFUS stimulation. The results demonstrated that the stimulation-induced motor response success rates in Piezo knockdown mice were lower compared to the control group (Piezo1 knockdown: 57.63% ± 14.62%, Piezo2 knockdown: 73.71% ± 13.10%, Control mice: 85.69% ± 10.23%). Both Piezo1 and Piezo2 knockdowns showed prolonged motor response times (Piezo1 knockdown: 0.62 ± 0.19 s, Piezo2 knockdown: 0.60 ± 0.13 s, Control mice: 0.44 ± 0.12 s) compared to controls. Additionally, Piezo knockdown animals subjected to tFUS showed reduced immunofluorescent c-Fos expression in the target area when measured in terms of cells per unit area compared to the control group. This in vivo study confirms the pivotal role of Piezo channels in tFUS-induced neuromodulation, highlighting their influence on motor response efficacy and timing. This study provides insights into the mechanistic underpinnings of noninvasive brain stimulation techniques and opens avenues for developing targeted therapies for neural disorders.

Low hepatic artery blood flow mediates NET extravasation through the regulation of PIEZO1/SRC signaling to induce biliary complications after liver transplantation.

Rationale: Biliary complications after liver transplantation persistently affect patient prognosis and graft survival. Neutrophil-mediated immune injury is an important factor leading to biliary injury. However, the mechanism by which neutrophils reach the periphery of the bile duct and further mediate bile duct injury is not fully understood. Methods: First, we obtained hepatic tissue samples from grafted rats subjected to warm and nonwarm ischemic injury. We constructed a protein map via proteomics and analyzed the correlations between neutrophil extracellular traps (NETs) and biliary injury. HuCCT1 cells were cocultured with NETs isolated from the peripheral blood of grafted rats in vitro to evaluate the role of NETs in bile duct injury. Next, we assessed NET extravasation through the PIEZO1/SRC pathway in liver samples from rats with liver grafts via proteomic analysis, immunohistochemical staining and immunofluorescence. Finally, we evaluated the correlations between hepatic arterial blood flow and the PIEZO1/SRC pathway in a liver graft model. Results: The results revealed a close correlation between NET formation by activated neutrophils and bile duct injury. Low hepatic arterial blood flow leads to NET extravasation through the activation of the mechanosensitive ion channel PIEZO1 and its downstream signaling events, including phosphorylation of tyrosine kinases sarcoma (SRC) protein. The extravasated NETs accumulate around the bile ducts and subsequently mediate biliary cell apoptosis. Verapamil was further used to increase hepatic artery blood flow to inhibit the PIEZO1/SRC axis, which reduced bile duct injury caused by extravasated NETs. Conclusions: Suppressing NET extravasation by increasing hepatic arterial blood flow is a potential strategy for the treatment of biliary complications after liver transplantation.

Nanoscale Magneto-mechanical-genetics of Deep Brain Neurons Reversing Motor Deficits in Parkinsonian Mice.

Here, we introduce the magneto-mechanical-genetic (MMG)-driven wireless deep brain stimulation (DBS) using magnetic nanostructures for therapeutic benefits in the mouse model of Parkinson's disease (PD). Electrical DBS of the subthalamic nucleus (STN) is an effective therapy for mitigating Parkinson's motor symptoms. However, its broader application is hampered by the requirement for implanted electrodes and the lack of anatomical and cellular specificity. Using the nanoscale magnetic force actuators (m-Torquer), which deliver torque force under rotating magnetic fields to activate pre-encoded Piezo1 ion channels on target neurons, our system enables wireless and STN-specific DBS without implants, addressing key unmet challenges in the DBS field. In both late- and early-stage PD mice, MMG-DBS significantly improved locomotor activity and motor balance by 2-fold compared to untreated PD mice. Moreover, MMG-DBS enabled sustained therapeutic effects. This approach provides a non-invasive and implant-free DBS with cellular targeting capability for the effective treatment of Parkinsonian symptoms.

Roles of the mechanosensitive ion channel Piezo1 in the renal podocyte injury of experimental hypertensive nephropathy.

Glomerular podocyte injury plays an essential role in proteinuria pathogenesis, a hallmark of chronic kidney disease, including hypertensive nephropathy. Although podocytes are susceptible to mechanical stimuli, their mechanotransduction pathways remain elusive. Piezo proteins, including Piezo1 and 2, are mechanosensing ion channels that mediate various biological phenomena. Although renal Piezo2 expression and its alteration in rodent dehydration and hypertension models have been reported, the role of Piezo1 in hypertensive nephropathy and podocyte injury is unclear. In this study, we examined Piezo1 expression and localization in the kidneys of control mice and in those of mice with hypertensive nephrosclerosis. Uninephrectomized, aldosterone-infused, salt-loaded mice developed hypertension, albuminuria, podocyte injury, and glomerulosclerosis. RNAscope in situ hybridization revealed that Piezo1 expression was enhanced in the podocytes, mesangial cells, and distal tubular cells of these mice compared to those of the uninephrectomized, vehicle-infused control group. Piezo1 upregulation in the glomeruli was accompanied by the induction of podocyte injury-related markers, plasminogen activator inhibitor-1 and serum/glucocorticoid regulated kinase 1. These changes were reversed by antihypertensive drug. Exposure of Piezo1-expressing cultured podocytes to mechanical stretch activated Rac1 and upregulated the above-mentioned markers, which was antagonized by the Piezo1 blocker grammostola mechanotoxin #4 (GsMTx4). Administration of Piezo1-specific agonist Yoda1 mimicked the effects of mechanical stretch, which was minimized by the Yoda1-specific inhibitor Dooku1 and Rac inhibitor. Rac1 was also activated in the above-mentioned hypertensive mice, and Rac inhibitor downregulated gene expression of podocyte injury-related markers in vivo. Our results suggest that Piezo1 plays a role in mechanical stress-induced podocyte injury.

Endothelial Response to the Combined Biomechanics of Vessel Stiffness and Shear Stress Is Regulated via Piezo1.

How endothelial cells sense and respond to dynamic changes in their biophysical surroundings as we age is not fully understood. Vascular stiffness is clearly a contributing factor not only in several cardiovascular diseases but also in physiological processes such as aging and vascular dementia. To address this gap, we utilized a microfluidic model to explore how substrate stiffness in the presence of shear stress affects endothelial morphology, senescence, proliferation, and inflammation. We also studied the role of mechanosensitive ion channel Piezo1 in endothelial responses under the combined effect of shear stress and substrate stiffness. To do so, we cultured endothelial cells inside microfluidic channels covered with fibronectin-coated elastomer with elastic moduli of 40 and 200 kPa, respectively, mimicking the stiffness of the vessel walls in young and aged arteries. The endothelial cells were exposed to atheroprotective and atherogenic shear stress levels of 10 and 2 dyn/cm2, respectively. Our findings show that substrate stiffness affects senescence under atheroprotective flow conditions and cytoskeleton remodeling, senescence, and inflammation under atherogenic flow conditions. Additionally, we found that the expression of Piezo1 plays a crucial role in endothelial adaptation to flow and regulation of inflammation under both atheroprotective and atherogenic shear stress levels. However, Piezo1 contribution to endothelial senescence was limited to the soft substrate and atheroprotective shear stress level. Overall, our study characterizes the response of endothelial cells to the combined effect of shear stress and substrate stiffness and reveals a previously unidentified role of Piezo1 in endothelial response to vessel stiffening, which potentially can be therapeutically targeted to alleviate endothelial dysfunction in aging adults.

Piezo1 Is Required for Myoblast Migration and Involves Polarized Clustering in Association with Cholesterol and GM1 Ganglioside.

A specific plasma membrane distribution of the mechanosensitive ion channel Piezo1 is required for cell migration, but the mechanism remains elusive. Here, we addressed this question using WT and Piezo1-silenced C2C12 mouse myoblasts and WT and Piezo1-KO human kidney HEK293T cells. We showed that cell migration in a cell-free area and through a porous membrane decreased upon Piezo1 silencing or deletion, but increased upon Piezo1 activation by Yoda1, whereas migration towards a chemoattractant gradient was reduced by Yoda1. Piezo1 organized into clusters, which were preferentially enriched at the front. This polarization was stimulated by Yoda1, accompanied by Ca2+ polarization, and abrogated by partial cholesterol depletion. Piezo1 clusters partially colocalized with cholesterol- and GM1 ganglioside-enriched domains, the proportion of which was increased by Yoda1. Mechanistically, Piezo1 activation induced a differential mobile fraction of GM1 associated with domains and the bulk membrane. Conversely, cholesterol depletion abrogated the differential mobile fraction of Piezo1 associated with clusters and the bulk membrane. In conclusion, we revealed, for the first time, the differential implication of Piezo1 depending on the migration mode and the interplay between GM1/cholesterol-enriched domains at the front during migration in a cell-free area. These domains could provide the optimal biophysical properties for Piezo1 activity and/or spatial dissociation from the PMCA calcium efflux pump.

Marginal effect of hydrostatic pressure on osteogenic differentiation of mesenchymal stem cells and its mechanism

Hydrostatic pressure (HP) is one of the important mechanical stimuli to maintain the structure and function of bone tissue. Under non-physiological conditions, abnormal increases or decreases in HP can lead to loss of bone tissue function. Although HP has been proven to affect osteogenic differentiation, the rules and mechanisms of its regulatory effect are still unclear. In this study, the HP showed a marginal effect on promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). The regulation of osteogenic differentiation by HP is closely related to the Piezo1 ion channel. On the one hand, the activity of mTOR is inhibited by HP through the CaMKK2/AMPKα/mTOR signaling axis, thereby promoting the osteogenic differentiation. On the other hand, the cell morphology is also regulated by HP and tends to be round, which is not conducive to osteogenesis. The two regulatory modes jointly control the ultimate osteogenic differentiation ability of BMSCs in a complex way. This study provides new insights into the mechanism by which HP regulates osteogenic differentiation, suggesting the need to consider the role of signaling cascades and physical changes simultaneously.

Higher-intensity ultrasound accelerates fracture healing via mechanosensitive ion channel Piezo1.

Osteoporosis-related fractures are a major public health problem. Mechanobiological stimulation utilizing low-intensity pulsed ultrasound (LIPUS) is the most widely accepted modality for accelerating fracture healing. However, recent evidence has demonstrated the ineffectiveness of LIPUS, and the biophysical mechanisms of ultrasound-induced bone formation also remain elusive. Here, we demonstrate that ultrasound at a higher intensity than LIPUS effectively accelerates fracture healing in a mouse osteoporotic fracture model. Higher-intensity ultrasound promoted chondrogenesis and hypertrophic differentiation of chondrocytes in the fracture callus. Higher-intensity ultrasound also increased osteoblasts and newly formed bone in the callus, resulting in accelerated endochondral ossification during fracture healing. In addition, we found that accelerated fracture healing by ultrasound exposure was attenuated when the mechanosensitive ion channel Piezo1 was inhibited by GsMTx4. Ultrasound-induced new bone formation in the callus was attenuated in fractured mice treated with GsMTx4. Similar results were also confirmed in a 3D osteocyte-osteoblast co-culture system, where osteocytic Piezo1 knockdown attenuated the expression of osteoblastic genes after ultrasound exposure. Together these results demonstrate that higher-intensity ultrasound than clinically used LIPUS can accelerate endochondral ossification after fractures. Furthermore, our results suggest that mechanotransduction via Piezo1 mediates ultrasound-stimulated fracture healing and bone formation.

Blood Flow Directs Yap/Taz-Mediated Transcriptional Regulation of Self-Renewal Programs to Control Developmental HSPC Expansion By Mechanical Stimulation of Piezo1

Hematopoietic stem and progenitor cells (HSPCs) emerge from artery-derived hemogenic endothelium (HE) in the vertebrate embryo during development. This process entails a cellular reprogramming from endothelial to hematopoietic gene signatures driven by the Runx1 transcription factor (TF), which is referred to as the endothelial-to-hematopoietic transition (EHT). A major goal of cellular therapeutics is to derive patient-specific HSPCs from iPSCs for clinical use, yet current differentiation protocols largely fail to recapitulate EHT to produce or expand long-lived multi-potent HSPCs in vitro suggesting an incomplete understanding of the in vivo regulatory cues. Physical forces of wall shear stress (WSS) and cyclic stretch (CS) produced by hemodynamic blood flow in embryos are one such cue required during EHT to generate HSPCs from HE, however the mechanisms by which these forces are sensed and converted into a “stemness” regulatory module remain undefined. Our previous work implicated the Hippo pathway TF YAP as essential for the maintenance, but not initiation, of the hematopoietic program in newly specified HE. Here, using scRNA-sequencing of sorted zebrafish trunk endothelial cells to capture all stages of EHT progression, we find that overexpression of YAP drives lipid metabolism, cell cycling, and propagation of a hematopoietic gene regulatory network (GRN) in the earliest specified HE cells. By employing a heat shock-inducible dominant negative YAP zebrafish line, we unmask a latent role for the YAP paralogue TAZ in hematopoiesis, which can promote CD41 + and Flk +/Myb + HSPC production upon reduced YAP function. YAP and TAZ initiate transcriptional responses downstream of mechanical stimuli and require DNA binding cofactors to regulate target genes. Surprisingly, luciferase assays in HEK293 cells demonstrated a potent synergistic effect of TAZ/RUNX1, but not YAP/RUNX1, in transcriptional regulation at RUNX enhancers. Finally, by pharmacologic and genetic manipulation, we identify the stretch-gated membrane ion channel Piezo1 as a potent regulator of CS-induced YAP/TAZ mechanotransduction in HE. Stimulation of zebrafish embryos with the Piezo1 small molecule agonist Yoda1 significantly increases HSPC number and YAP target gene expression in a YAP-dependent fashion. A similar modulation of blood and YAP target genes in human iPSC-derived CD34+ HE cells is seen with Yoda1, suggesting that this stretch-Piezo1-YAP axis can be chemically tuned in vitro to enhance HSPC differentiation. These results illuminate molecular details of how YAP/TAZ directs the production of HSPCs from HE in response to mechanical cues, and have broader implications for alternate regulatory effects of mechanically-stimulated Hippo TFs depending on the transcriptional milieu in cell-type specific contexts.

Immunolocalization of the mechanogated ion channels PIEZO1 and PIEZO2 in human and mouse dental pulp and periodontal ligament.

PIEZO1 and PIEZO2 are essential components of mechanogated ion channels, which are required for mechanotransduction and biological processes associated with mechanical stimuli. There is evidence for the presence of PIEZO1 and PIEZO2 in teeth and periodontal ligaments, especially in cell lines and mice, but human studies are almost nonexistent. Decalcified permanent human teeth and mouse molars were processed for immunohistochemical detection of PIEZO1 and PIEZO2. Confocal laser microscopy was used to examine the co-localization of PIEZO 1 and PIEZO2 with vimentin (a marker of differentiated odontoblasts) in human teeth. In the outer layer of the human dental pulp, abundant PIEZO1- and PIEZO2-positive cells were found that had no odontoblast morphology and were vimentin-negative. Based on their morphology, location, and the absence of vimentin positivity, they were identified as dental pulp stem cells or pre-odontoblasts. However, in mice, PIEZO1 and PIEZO2 were ubiquitously detected and colocalized in odontoblasts. Intense immunoreactivity of PIEZO1 and PIEZO2 has been observed in human and murine periodontal ligaments. Our findings suggest that PIEZO1 and PIEZO2 may be mechanosensors/mechanotransducers in murine odontoblasts, as well as in the transmission of forces by the periodontal ligament in humans and mice.