Mechanotransduction Processes Research Articles

Systematic Review on Working Mechanisms of Signaling Pathways in Fibrosis During Shockwave Therapy.

Fibrosis is characterized by scarring and hardening of tissues and organs. It can affect every organ system, and so could result in organ failure due to the accumulation of extracellular matrix proteins. Previous studies suggest that mechanical forces (such as shockwave therapy, SWT) initiate a process of mechanotransduction and thus could regulate fibrosis. Nevertheless, it is largely unexamined which pathways are exactly involved in the application of SWT and can regulate fibrosis. The present article seeks to elucidate the underlying effect of SWT on fibrosis. Evidence shows that SWT activates macrophage activity, fibroblast activity, collagen amount and orientation and apoptosis, which ultimately lead to an adaptation of inflammation, proliferation, angiogenesis and apoptosis. The included articles reveal that other proteins and pathways can be activated depending on the energy levels and frequency of SWT. These findings demonstrate that SWT has beneficial effects on fibrosis by influencing the proteins and pathways. Based on these data, which highlights the underlying mechanisms, we can make preliminary conclusions about the treatment modalities of SWT in scar formation, such as the energy levels and frequencies that are necessary to prevent or treat fibrotic tissue.

Toward understanding the brain tissue behavior due to preconditioning: an experimental study and RVE approach.

Brain tissue under preconditioning, as a complex issue, refers to repeated loading-unloading cycles applied in mechanical testing protocols. In previous studies, only the mechanical behavior of the tissue under preconditioning was investigated; However, the link between macrostructural mechanical behavior and microstructural changes in brain tissue remains underexplored. This study aims to bridge this gap by investigating bovine brain tissue responses both before and after preconditioning. We employed a dual approach: experimental mechanical testing and computational modeling. Experimental tests were conducted to observe microstructural changes in mechanical behavior due to preconditioning, with a focus on axonal damage. Concurrently, we developed multiscale models using statistically representative volume elements (RVE) to simulate the tissue's microstructural response. These RVEs, featuring randomly distributed axonal fibers within the extracellular matrix, provide a realistic depiction of the white matter microstructure. Our findings show that preconditioning induces significant changes in the mechanical properties of brain tissue and affects axonal integrity. The RVE models successfully captured localized stresses and facilitated the microscopic analysis of axonal injury mechanisms. These results underscore the importance of considering both macro and micro scales in understanding brain tissue behavior under mechanical loading. This comprehensive approach offers valuable insights into mechanotransduction processes and improves the analysis of microstructural phenomena in brain tissue.

Strategic Approaches in Generation of Robust Microphysiological 3D Musculoskeletal Tissue System

AbstractSkeletal muscle plays a vital role in maintaining the body's shape and regulating various physiological processes. Its function is influenced by a multitude of factors. Given the lack of uniformity in prior research regarding the size and placement of structural pillars within the chip, as well as the choice of cell‐laden hydrogel components with various densities of extracellular matrix, in different gelation times, and cell densities, a meticulous investigation is conducted to enhance the robustness of 3D in vitro musculoskeletal tissues. This study provides guidance on how to optimize the design parameters of skeletal muscle‐on‐a‐chip and hydrogel recipe by evaluating the impact of design elements and hydrogel fabrication conditions on tissue formation and musculoskeletal differentiation. This research reports the direct evidence of mechanical properties of hydrogels are critical in influencing cellular differentiation and tissue functionality through the process of mechanotransduction. The study highlights the importance of standardizing experimental conditions in 3D in vitro musculoskeletal research, and presents a validated framework as a foundation to aid in the development of functional musculoskeletal tissue for clinical and research applications, including disease modeling and regenerative therapies.

Understanding mechanotransduction in the distal colon and rectum via multiscale and multimodal computational modeling

Visceral pain in the large bowel is a defining symptom of irritable bowel syndrome (IBS) and the primary reason that patients visit gastroenterologists. This pain is reliably triggered by mechanical distension of the distal colon and rectum (colorectum). Consequently, the process of mechanotransduction by sensory afferents, responsible for translating mechanical colorectal stimuli into neural action potentials, plays a central role in IBS-related bowel pain. In this study, we aim to enhance our understanding of colorectal mechanotransduction by combining experimental findings in colorectal biomechanics and afferent neural encoding within a comprehensive computational simulation framework. To achieve this, we implemented a three-layered, fiber-reinforced finite element model that accurately replicates the nonlinear, heterogeneous, and anisotropic mechanical characteristics of the mouse colorectum. This model facilitates the computation of local mechanical stresses and strains around individual afferent endings, which have diameters on the micron-scale. We then integrated a neural membrane model to simulate the encoding of action potentials by afferent nerves in response to microscopic stresses and strains along the afferent endings. Our multiscale simulation framework enables the assessment of three hypotheses regarding the mechanical gating of action potential generation: (1) axial stress dominates mechanical gating of mechanosensitive channels, (2) both axial and circumferential stresses contribute, and (3) membrane shear stress dominates. Additionally, we explore how the orientation of afferent endings impacts neural encoding properties. This computational framework not only allows for the virtual investigation of colorectal mechanotransduction in the context of prolonged visceral hypersensitivity but can also guide the development of new experimental studies aimed at uncovering the neural and biomechanical mechanisms underlying IBS-related bowel pain.

Mechanosensitive Piezo channels and their potential roles in peripheral auditory perception

AbstractHearing sound and responding to external and internal mechanical stimuli requires specific proteins as mechanotransducers that convert mechanical forces into biological signals. However, our understanding of the mechanotransduction process in the inner ear is still incomplete. Mechanically activated ion channels, PIEZO1 and PIEZO2, are widely distributed throughout the body and play essential roles. Recent studies have discovered that Piezo channels are expressed in inner ear hair cells, suggesting their potential involvement in auditory perception. This review summarizes the existing discoveries about the Piezo channels, including their structure, mechanogating mechanisms, and general physiological roles, explicitly focusing on Piezo channels in the auditory systems. Piezo channels play roles in ultrasound perception, generation of anomalous current, hair cell development, and potentially in the normal mechanoelectrical transduction process of hair cells. Collectively, this review aims to provide a new perspective on the Piezo channel and its potential roles in auditory perception.

Flexible and multi-functional three-dimensional scaffold based on enokitake-like Au nanowires for real-time monitoring of endothelial mechanotransduction

Endothelial cells are sensitive to mechanical force and can convert it into biochemical signals to trigger mechano-chemo-transduction. Although conventional techniques have been used to investigate the subsequent modifications of cellular expression after mechanical stimulation, the in situ and real-time acquiring the transient biochemical information during mechanotransduction process remains an enormous challenge. In this work, we develop a flexible and multi-functional three-dimensional conductive scaffold that integrates cell growth, mechanical stimulation, and electrochemical sensing by in situ growth of enokitake-like Au nanowires on a three-dimensional porous polydimethylsiloxane substrate. The conductive scaffold possesses stable and desirable electrochemical sensing performance toward nitric oxide under mechanical deformation. The prepared e-AuNWs/CC/PDMS scaffold exhibits a good electrocatalytic ability to NO with a linear range from 2.5 nM to 13.95 μM and a detection limit of 8 nM. Owing to the excellent cellular compatibility, endothelial cells can be cultured directly on the scaffold and the real-time inducing and recording of nitric oxide secretion under physiological and pathological conditions were achieved. This work renders a reliable sensing platform for real-time monitoring cytomechanical signaling during endothelial mechanotransduction and is expected to promote other related biological investigations based on three-dimensional cell culture.

Modulating cell stiffness for improved vascularization: leveraging the MIL-53(fe) for improved interaction of titanium implant and endothelial cell

Vascularization plays a significant role in promoting the expedited process of bone regeneration while also enhancing the stability and viability of artificial bone implants. Although titanium alloy scaffolds were designed to mimic the porous structure of human bone tissues to facilitate vascularization in bone repair, their biological inertness restricted their broader utilization. The unique attribute of Metal-organic framework (MOF) MIL-53(Fe), known as “breathing”, can facilitate the efficient adsorption of extracellular matrix proteins and thus provide the possibility for efficient interaction between scaffolds and cell adhesion molecules, which helps improve the bioactivity of the titanium alloy scaffolds. In this study, MIL-53(Fe) was synthesized in situ on the scaffold after hydrothermal treatment. The MIL-53(Fe) endowed the scaffold with superior protein absorption ability and preferable biocompatibility. The scaffolds have been shown to possess favorable osteogenesis and angiogenesis inducibility. It was indicated that MIL-53(Fe) modulated the mechanotransduction process of endothelial cells and induced increased cell stiffness by promoting the adsorption of adhesion-mediating extracellular matrix proteins to the scaffold, such as laminin, fibronectin, and perlecan et al., which contributed to the activation of the endothelial tip cell phenotype at sprouting angiogenesis. Therefore, this study effectively leveraged the intrinsic “breathing” properties of MIL-53 (Fe) to enhance the interaction between titanium alloy scaffolds and vascular endothelial cells, thereby facilitating the vascularization inducibility of the scaffold, particularly during the sprouting angiogenesis phase. This study indicates that MIL-53(Fe) coating represents a promising strategy to facilitate accelerated and sufficient vascularization and uncovers the scaffold-vessel interaction from a biomechanical perspective.Graphical

Optimizing Uterine Synchronization Analysis in Pregnancy and Labor Through Window Selection and Node Optimization

1) Introduction: Preterm labor (PL) has globally become the leading cause of death in children under the age of 5 years. One of the most significant keys to preventing preterm labor is its early detection. 2) Objectives: The primary objectives of this study are to address the problem of PL by providing a new approach by analyzing the electrohysterographic (EHG) signals, which are recorded on the mother's abdomen during labor and pregnancy. 3) Methods: The EHG signal reflects the electrical activity that induces the mechanical contraction of the myometrium. Because EHGs are known to be non-stationary signals, and because we anticipate connectivity to alter during contraction (due to electrical diffusion and the mechanotransduction process), we applied the windowing approach on real signals to identify the best windows and the best nodes with the most significant data to be used for classification. The suggested pipeline includes: i) dividing the 16 EHG signals that are recorded from the abdomen of pregnant women in N windows; ii) apply the connectivity matrices on each window; iii) apply the Graph theory-based measures on the connectivity matrices on each window; iv) apply the consensus Matrix on each window in order to retrieve the best windows and the best nodes. Following that, several neural network and machine learning methods are applied to the best windows and best nodes to categorize pregnancy and labor contractions, based on the different input parameters (connectivity method alone, connectivity method plus graph parameters, best nodes, all nodes, best windows, all windows). 4) Results: Results showed that the best nodes are nodes 8, 9, 10, 11, and 12; while the best windows are 2, 4, and 5. The classification results obtained by using only these best nodes are better than when using the whole nodes. The results are always better when using the full burst, whatever the chosen nodes. 5) Conclusion: The windowing approach proved to be an innovative technique that can improve the differentiation between labor and pregnancy EHG signals.

Creating a bionic scaffold via light-curing liquid crystal ink to reveal the role of osteoid-like microenvironment in osteogenesis

Osteoid plays a crucial role in directing cell behavior and osteogenesis through its unique characteristics, including viscoelasticity and liquid crystal (LC) state. Thus, integrating osteoid-like features into 3D printing scaffolds proves to be a promising approach for personalized bone repair. Despite extensive research on viscoelasticity, the role of LC state in bone repair has been largely overlooked due to the scarcity of suitable LC materials. Moreover, the intricate interplay between LC state and viscoelasticity in osteogenesis remains poorly understood. Here, we developed innovative hydrogel scaffolds with osteoid-like LC state and viscoelasticity using digital light processing with a custom LC ink. By utilizing these LC scaffolds as 3D research models, we discovered that LC state mediates high protein clustering to expose accessible RGD motifs to trigger cell-protein interactions and osteogenic differentiation, while viscoelasticity operates via mechanotransduction pathways. Additionally, our investigation revealed a synergistic effect between LC state and viscoelasticity, amplifying cell-protein interactions and osteogenic mechanotransduction processes. Furthermore, the interesting mechanochromic response observed in the LC hydrogel scaffolds suggests their potential application in mechanosensing. Our findings shed light on the mechanisms and synergistic effects of LC state and viscoelasticity in osteoid on osteogenesis, offering valuable insights for the biomimetic design of bone repair scaffolds.

Mechanobiological Modulation of In Vitro Astrocyte Reactivity Using Variable Gel Stiffness.

After traumatic brain injury, the brain extracellular matrix undergoes structural rearrangement due to changes in matrix composition, activation of proteases, and deposition of chondroitin sulfate proteoglycans by reactive astrocytes to produce the glial scar. These changes lead to a softening of the tissue, where the stiffness of the contusion "core" and peripheral "pericontusional" regions becomes softer than that of healthy tissue. Pioneering mechanotransduction studies have shown that soft substrates upregulate intermediate filament proteins in reactive astrocytes; however, many other aspects of astrocyte biology remain unclear. Here, we developed a platform for the culture of cortical astrocytes using polyacrylamide (PA) gels of varying stiffness (measured in Pascal; Pa) to mimic injury-related regions in order to investigate the effects of tissue stiffness on astrocyte reactivity and morphology. Our results show that substrate stiffness influences astrocyte phenotype; soft 300 Pa substrates led to increased GFAP immunoreactivity, proliferation, and complexity of processes. Intermediate 800 Pa substrates increased Aggrecan+, Brevican+, and Neurocan+ astrocytes. The stiffest 1 kPa substrates led to astrocytes with basal morphologies, similar to a physiological state. These results advance our understanding of astrocyte mechanotransduction processes and provide evidence of how substrates with engineered stiffness can mimic the injury microenvironment.

Focal adhesion and actin orientation regulated by cellular geometry determine stem cell differentiation via mechanotransduction

Tuning cell adhesion geometry can affect cytoskeleton organization and the distribution of cytoskeleton forces, which play critical roles in controlling cell functions. To elucidate the geometrical relationship with cytoskeleton force distribution, it is necessary to control cell morphology. In this study, a series of dextral vortex micropatterns were prepared to precisely control cell morphology for investigating the influence of the curvature degree of adhesion curves on intracellular force distribution and stem cell differentiation at a sub-cellular level. Peripherial actin filaments of micropatterned cells were assembled along the adhesion curves and showed different orientations, filament thicknesses and densities. Focal adhesion and cytoskeleton force distribution were dependent on the curvature degree. Intracellular force distribution was also regulated by adhesion curves. The cytoskeleton and force distribution affected the osteogenic differentiation of mesenchymal stem cells through a YAP/TAZ-mediated mechanotransduction process. Thus, regulation of cell adhesion curvature, especially at cytoskeletal filament level, is critical for cell function manipulation. Statement of significanceIn this study, a series of dextral micro-vortexes were prepared and used for the culture of human mesenchymal stem cells (hMSCs) to precisely control adhesive curvatures (0°, 30°, 60°, and 90°). The single MSCs on the micropatterns had the same size and shape but showed distinct focal adhesion (FA) and cytoskeleton orientations. Cellular nanomechanics were observed to be correlated with the curvature degrees, subsequently influencing nuclear morphological features. As a consequence, the localization of the mechanotransduction sensor and activator-YAP/TAZ was affected, influencing osteogenic differentiation. The results revealed the pivotal role of adhesive curvatures in the manipulation of stem cell differentiation via the machanotransduction process, which has rarely been investigated.

Red blood cell flickering activity locally controlled by holographic optical tweezers

Red blood cells possess a singular mechanobiology, enabling efficient navigation through capillaries smaller than their own size. Their plasma membrane exhibits non-equilibrium shape fluctuation, often reported as enhanced flickering activity. Such active membrane motion is propelled by motor proteins that mediate interactions between the spectrin skeleton and the lipid bilayer. However, modulating the flickering in living red blood cells without permanently altering their mechanical properties represents a significant challenge. In this study, we developed holographic optical tweezers to generate a force field distributed along the equatorial membrane contour of individual red blood cells. In free-standing red blood cells, we observed heterogeneous flickering activity, attributed to localized membrane kickers. By employing holographic optical forces, these active kickers can be selectively halted under minimal invasion. Our findings shed light on the dynamics of membrane flickering and established a manipulation tool that could open new avenues for investigating mechanotransduction processes in living cells.

Differentiation and functioning of the lateral line organ in zebrafish require Smpx activity

The small muscle protein, X-linked (SMPX) gene encodes a cytoskeleton-associated protein, highly expressed in the inner ear hair cells (HCs), possibly regulating auditory function. In the last decade, several mutations in SMPX have been associated with X-chromosomal progressive non syndromic hearing loss in humans and, in line with this, Smpx-deficient animal models, namely zebrafish and mouse, showed significant impairment of inner ear HCs development, maintenance, and functioning. In this work, we uncovered smpx expression in the neuromast mechanosensory HCs of both Anterior and Posterior Lateral Line (ALL and PLL, respectively) of zebrafish larvae and focused our attention on the PLL. Smpx was subcellularly localized throughout the cytoplasm of the HCs, as well as in their primary cilium. Loss-of-function experiments, via both morpholino-mediated gene knockdown and CRISPR/Cas9 F0 gene knockout, revealed that the lack of Smpx led to fewer properly differentiated and functional neuromasts, as well as to a smaller PLL primordium (PLLp), the latter also Smpx-positive. In addition, the kinocilia of Smpx-deficient neuromast HCs appeared structurally and numerically altered. Such phenotypes were associated with a significant reduction in the mechanotransduction activity of the neuromast HCs, in line with their positivity for Smpx. In summary, this work highlights the importance of Smpx in lateral line development and, specifically, in proper HCs differentiation and/or maintenance, and in the mechanotransduction process carried out by the neuromast HCs. Because lateral line HCs are both functionally and structurally analogous to the cochlear HCs, the neuromasts might represent an invaluable—and easily accessible—tool to dissect the role of Smpx in HCs development/functioning and shed light on the underlying mechanisms involved in hearing loss.

A modified motor-clutch model reveals that neuronal growth cones respond faster to soft substrates.

Neuronal growth cones sense a variety of cues including chemical and mechanical ones to establish functional connections during nervous system development. Substrate-cytoskeletal coupling is an established model for adhesion-mediated growth cone advance; however, the detailed molecular and biophysical mechanisms underlying the mechanosensing and mechanotransduction process remain unclear. Here, we adapted a motor-clutch model to better understand the changes in clutch and cytoskeletal dynamics, traction forces, and substrate deformation when a growth cone interacts with adhesive substrates of different stiffnesses. Model parameters were optimized using experimental data from Aplysia growth cones probed with force-calibrated glass microneedles. We included a reinforcement mechanism at both motor and clutch level. Furthermore, we added a threshold for retrograde F-actin flow that indicates when the growth cone is strongly coupled to the substrate. Our modeling results are in strong agreement with experimental data with respect to the substrate deformation and the latency time after which substrate-cytoskeletal coupling is strong enough for the growth cone to advance. Our simulations show that it takes the shortest time to achieve strong coupling when substrate stiffness was low at 4 pN/nm. Taken together, these results suggest that Aplysia growth cones respond faster and more efficiently to soft than stiff substrates.

How are cell and tissue structure and function influenced by gravity and what are the gravity perception mechanisms?

Progress in mechanobiology allowed us to better understand the important role of mechanical forces in the regulation of biological processes. Space research in the field of life sciences clearly showed that gravity plays a crucial role in biological processes. The space environment offers the unique opportunity to carry out experiments without gravity, helping us not only to understand the effects of gravitational alterations on biological systems but also the mechanisms underlying mechanoperception and cell/tissue response to mechanical and gravitational stresses. Despite the progress made so far, for future space exploration programs it is necessary to increase our knowledge on the mechanotransduction processes as well as on the molecular mechanisms underlying microgravity-induced cell and tissue alterations. This white paper reports the suggestions and recommendations of the SciSpacE Science Community for the elaboration of the section of the European Space Agency roadmap “Biology in Space and Analogue Environments” focusing on “How are cells and tissues influenced by gravity and what are the gravity perception mechanisms?” The knowledge gaps that prevent the Science Community from fully answering this question and the activities proposed to fill them are discussed.

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.

Bioactive prosthesis interface compositing variable-stiffness hydrogels regulates stem cells fates to facilitate osseointegration through mechanotransduction

Fluid hydrogel is proper to be incorporated with rigid porous prosthesis interface, acting as a soft carrier to support cells and therapeutic factors, to enhance osseointegration. In the previous study, we innovatively utilized self-healing supramolecular hydrogel as 3D cell culture platform to incorporate with 3D printed porous titanium alloy scaffold, constructing a novel bioactive interface. However, the concrete relationship and mechanism of hydrogel stiffness influencing cellular behaviors of bone marrow mesenchymal stem cells (BMSCs) within the interface are still inconclusive. Herein, we synthesized a series of supramolecular hydrogels with variable stiffness as extracellular matrix (ECM) to enhance the osseointegration of 3D printed prosthesis interface. BMSCs exposed to stiff hydrogel received massive environmental mechanical stimulations, subsequently transducing biophysical cues into biochemical signal through mechanotransduction process. The mRNA-sequencing analysis revealed that the activated FAK-MAPK pathway played significant roles in promoting osteogenic differentiation, thus contributing to a strong osseointegration. Our work preliminarily demonstrated the relationship of ECM stiffness and osteogenic differentiation trend of BMSCs, and optimized stiffness of hydrogel within a certain range benefitting for osteogenic differentiation and prosthesis interface osseointegration, providing a valuable insight into the development of orthopaedic implants equipped with osteogenic mechanotransduction ability.

Unveiling the Role of Mechanical Microenvironment in Hepatocellular Carcinoma: Molecular Mechanisms and Implications for Therapeutic Strategies.

Hepatocellular carcinoma (HCC) is the sixth most common cancer in the world and the third leading cause of cancer deaths globally. More than 80% of HCC patients have a background of fibrosis or cirrhosis, which leads to changes in physical factors in tumor microenvironment (TME), such as increased stiffness, solid stress, fluid stresses and structural alterations in the extracellular matrix (ECM). In the past, the focus of cancer research has predominantly been on genetic and biochemical factors in the TME, and the critical role of physical factors has often been overlooked. Recent discoveries suggest these unique physical signals are converted into biochemical signals through a mechanotransduction process that influences the biological behavior of tumor cells and stromal cells. This process facilitates the occurrence and progression of tumors. This review delves into the alterations in the mechanical microenvironment during the progression of liver fibrosis to HCC, the signaling pathways activated by physical signals, and the effects on both tumor and mesenchymal stromal cells. Furthermore, this paper summarizes and discusses the therapeutic options for targeting the mechanical aspects of the TME, offering valuable insights for future research into novel therapeutic avenues against HCC and other solid tumors.

Construction and operation of high-resolution magnetic tape head tweezers for measuring single-protein dynamics under force.

Mechanical forces are critical to protein function across many biological contexts-from bacterial adhesion to muscle mechanics and mechanotransduction processes. Hence, understanding how mechanical forces govern protein activity has developed into a central scientific question. In this context, single-molecule magnetic tweezers has recently emerged as a valuable experimental tool, offering the capability to measure single proteins over physiologically relevant forces and timescales. In this chapter, we present a detailed protocol for the assembly and operation of our magnetic tape head tweezers instrument, specifically tailored to investigate protein dynamics. Our instrument boasts a simplified microscope design and incorporates a magnetic tape head as the force-generating apparatus, facilitating precise force control and enhancing its temporal stability, enabling the study of single protein mechanics over extended timescales spanning several hours or even days. Moreover, its straightforward and cost-effective design ensures its accessibility to the wider scientific community. We anticipate that this technique will attract widespread interest within the growing field of mechanobiology and expect that this chapter will provide facilitated accessibility to this technology.

Targeting YAP/TAZ mechanosignaling to ameliorate stiffness-induced Schlemm's canal cell pathobiology.

Pathological alterations in the biomechanical properties of the Schlemm's canal (SC) inner wall endothelium and its immediate vicinity are strongly associated with ocular hypertension in glaucoma due to decreased outflow facility. Specifically, the underlying trabecular meshwork is substantially stiffer in glaucomatous eyes compared with that from normal eyes. This raises the possibility of a critical involvement of mechanotransduction processes in driving SC cell dysfunction. Yes-associated protein (YAP) has emerged as a key contributor to glaucoma pathogenesis. However, the molecular underpinnings of SC cell mechanosignaling via YAP and transcriptional coactivator with PDZ-binding motif (TAZ) in response to glaucomatous extracellular matrix (ECM) stiffening are not well understood. Using a novel biopolymer hydrogel that facilitates dynamic and reversible stiffness tuning, we investigated how ECM stiffening modulates YAP/TAZ activity in primary human SC cells, and whether disruption of YAP/TAZ mechanosignaling attenuates SC cell pathobiology and increases ex vivo outflow facility. We demonstrated that ECM stiffening drives pathologic YAP/TAZ activation and cytoskeletal reorganization in SC cells, which was fully reversible by matrix softening in a distinct time-dependent manner. Furthermore, we showed that pharmacologic or genetic disruption of YAP/TAZ mechanosignaling abrogates stiffness-induced SC cell dysfunction involving altered cytoskeletal and ECM remodeling. Finally, we found that perfusion of the clinically used, small molecule YAP/TAZ inhibitor verteporfin (without light activation) increases ex vivo outflow facility in normal mouse eyes. Collectively, our data provide new evidence for a pathologic role of aberrant YAP/TAZ mechanosignaling in SC cell dysfunction and suggest that YAP/TAZ inhibition has therapeutic value for treating ocular hypertension in glaucoma.NEW & NOTEWORTHY Pathologically altered biomechanical properties of the Schlemm's canal (SC) inner wall microenvironment were recently validated as the cause for increased outflow resistance in ocular hypertensive glaucoma. However, the involvement of specific mechanotransduction pathways in these disease processes is largely unclear. Here, we demonstrate that Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) are central regulators of glaucoma-like SC cell dysfunction in response to extracellular matrix stiffening and that targeted disruption of YAP/TAZ mechanosignaling attenuates SC cell pathobiology and enhances outflow function.