Role In Mechanotransduction Research Articles

The T‐type Voltage Gated Calcium Channel Cav3.2 is Important for Enteroendocrine Cell Mechanotransduction

BACKGROUNDEnteroendocrine cells (EECs) in the gastrointestinal (GI) epithelium are specialized sensors of luminal chemicals and forces. Upon stimulation, EECs release a variety of neurotransmitters and hormones, but cellular mechanisms of excitation‐secretion coupling are poorly understood. We recently discovered that a population of EECs expresses the mechanosensitive ion channel Piezo2, which is necessary for force‐dependent intracellular Ca2+ increase and serotonin release. However, the molecular mechanism that links Piezo2 and serotonin release is unknown. In other sensory systems, the Cacna1h‐encoded t‐type voltage‐gated calcium channel Cav3.2 is important in mechanotransduction for linking mechanical activation with neurotransmitter release.AIMto determine if Cav3.2 is expressed and functionally relevant in EECs.METHODSQuantitative RT‐PCR for Cacna1h mRNA expression was evaluated in colon epithelium of control and NeuroD1 Cre x RiboTag mice, in which actively transcribed mRNA specifically from EECs is tagged with hemagglutinin (HA). NeuroD1 Cre x tdTomato colon cross‐sections were fixed and immunolabeled with anti‐Cav3.2 antibody. Single primary NeuroD1 cre x GCaMP5 EECs were mechanically stimulated and Cav3.2 role in mechanotransduction was tested by pharmacology (Ca2+ free, nickel, mibefradil, ω‐agatoxin IVA) and Cacna1h siRNA knockdown.RESULTSMurine colon epithelium expressed Cav3.2 mRNA. In NeuroD1 Cre x RiboTag mouse, HA co‐localized with EEC marker Chromogranin A (Chga). NeuroD1+ EEC ribosomal trapped mRNA was enriched for markers of epithelium (Vil1), EEC (Chga), and enriched 7.0‐fold for Cav3.2 (n=3, p<0.05 compared to whole epithelium). Immunohistochemistry in NeuroD1 Cre x tdTomato showed that Cav3.2 co‐localized specifically with tdTomato (NeuroD1+) EECs. In primary NeuroD1+ EECs, Cav3.2 inhibition by all pharmacologic agents and siRNA knockdown inhibited mechanically induced Ca2+ response (96±1% for Ca2+ free, 71±9% nickel, 61±6% mibefradil, 80±4% ω‐agatoxin IVA and 96±1% Cav3.2 siRNA, n=3–18, p<0.05 compared to control untreated cells or cells treated with non‐targeted siRNA).CONCLUSIONSCav3.2 is densely and specifically expressed in mouse colon EECs and it is important for regulating Ca2+ in EEC mechanotransduction. Future studies will determine how Cav3.2 channels contribute to EEC electrical excitability, excitation‐secretion coupling, and ultimately EEC Cav3.2 roles in GI physiology.Support or Funding InformationSupported by NIH DK106456, NIH DK084567, and AGA RSAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Botox-induced muscle paralysis alters intracortical porosity and osteocyte lacunar density in skeletally mature rats.

Reduced mechanical loading can lead to disuse osteoporosis, resulting in bone fragility. Disuse models report macroscopic bone loss due to muscle inactivity and immobilization, yet only recently has there been quantification of the effects of disuse on the vascular pores and osteocyte network, which are believed to play an important role in mechanotransduction via interstitial fluid flow. The goal of this study was to perform a high-resolution analysis of the effects of muscle inactivity on intracortical porosity and osteocyte lacunar density in skeletally mature rats. Muscle paralysis was induced in 20-week-old female Sprague Dawley rats by injection of botulinum neurotoxin. Rats were injected in the right hindlimb muscles with either Botox (BTX, n = 8) or saline solution (CTRL, n = 8), with a third group used as baseline controls (n = 8). Four weeks after injection, Botox caused a ∼60% reduction in hindlimb muscle mass. High-resolution micro-CT analysis showed that Botox-induced muscle paralysis increased vascular canal porosity and reduced osteocyte lacunar density within the tibial metaphysis cortex. Cortical thickness and other areal properties were diminished in the proximal tibial metaphysis, whereas no differences were found in the mid-diaphysis. Within the BTX group, the injected limbs showed a lower cancellous bone volume fraction relative to the contralateral limb. These results indicate that diminished muscle activity alters the vascular canal porosity and osteocyte lacunar density in cortical bone, which could alter interstitial fluid flow, affecting molecular transport and the transmission of mechanical signals to osteocytes. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Thy-1 as an Integrator of Diverse Extracellular Signals.

Thy-1 was discovered over 50 years ago, and in that time investigators from a broad variety of fields have described numerous and heterogeneous biological functions of Thy-1 in multiple contexts. As an outwardly facing cell surface molecule, it is well positioned to receive extracellular signals; previously reviewed studies have confirmed an important role in cell-cell and cell-matrix adhesion, cell migration, and regulation of outside-in signaling. More recent studies reviewed here expand the repertoire of Thy-1 effects on signaling pathways, and reveal novel roles in mechanotransduction, cellular differentiation, viral entry, and extracellular vesicle binding and internalization. All of these studies contribute to understanding Thy-1 as a context-dependent integrator of a diverse range of extracellular information, and provide impetus for further studies, some of which are suggested here.

Evolution of Stresses at Cell-Gel Interfaces during Confined Interfacial Migration

Cell migration at the interface of two substrates under confinement holds clinical significance as it is instrumental in morphogenesis and wound healing but has been insufficiently studied. The adhesions at the cell-gel interface are instrumental in deciding the fate of cell motility, viability and morphology. However, experiments have not been able to shed light into the evolution of stresses within the cells and at the interfaces. Therefore, a better understanding of the mechanics involved in such a process is necessary. The various components of the cell and both the gels were modelled as viscoelastic systems. We used finite element modelling to study the evolution of stresses and deformation within the cell and at the cell-gel interfaces. The cell is attached to an underlying soft gel substrate by focal adhesions (FAs), whereas, no adhesions are formed at the cell-stiff gel interface. The asymmetry in adhesion at the two cell-gel interfaces leads to an asymmetric stress state of the cell with relatively larger stress developing at the interface with stiff gel indicating higher actin density at this interface than at that with soft gel. This is validated by prior experiments. The location of FAs is found to play a decisive role in mechanotransduction as the FAs close to the region of protrusion are maximally stressed during motion but those farther away are scarcely engaged, which has also been demonstrated in prior experiments. An understanding of the stress evolution within the cell undergoing interfacial migration and at the interface can be potentially used to design wound healing tissue adhesives.

Regenerative rehabilitation: The role of mechanotransduction in orthopaedic regenerative medicine.

Regenerative rehabilitation is an emerging area of investigation that seeks to integrate regenerative medicine with rehabilitation medicine. It is based on the realization that combining these two areas of medicine at an early stage of treatment will produce a better clinical outcome than the traditional linear approach of first administering the elements of regeneration followed, after a delay, by rehabilitation. Indeed, in certain settings, a case can be made for initiating rehabilitation protocols before starting regenerative intervention. This review summarizes the contents of a workshop held during the 2018 annual meeting of the Orthopaedic Research Society. It introduced the concept of regenerative rehabilitation and then provided two orthopaedic examples drawn from the domains of cartilage repair and bone healing. Rehabilitation medicine can supply a variety of physical stimuli, including electrical stimulation, thermal stimulation and mechanical stimulation. Of these, mechanical stimulation has the most obvious relevance to orthopaedics. The mechano-responsiveness of cartilage and bone has been known for a long time, but is poorly understood and has led to only limited clinical application. Improved bioreactor designs that allow multi-axial loading enable new insights into the responsiveness of chondrocytes and chondroprogenitor cells to specific types of load, especially shear. Recent studies on the mechanobiology of bone healing show that modulating the mechanical environment of an experimental osseous lesion by a process of "Reverse Dynamization" soon after injury considerably enhances healing. Future studies are needed to probe the molecular mechanisms responsible for these phenomena and to translate these findings into clinical practice. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1263-1269, 2019.

Back to the Light Side: The Role of Mechanotransduction in the Paradoxical Response to Checkpoint Inhibitors in Cancer Patients.

A growing number of studies and case series point to a dark side of immune checkpoint inhibitors, as they may cause rapid tumor growth with potentially deleterious effects. The pathophysiological mechanism of hyper-progressive disease (HPD) is still unknown; in addition, there are no reliable predictive biomarkers that facilitate the process of patient selection for immunomodulatory antibody-based therapy. The proposed model attempts to reveal the mechanism of such paradoxical response, in a subset of patients receiving anti-PD1/PD-L1 immunotherapy, depending on the biomechanistic properties of the crystal structure of PD-1 protein. PD-1 can exhibit a signaling pattern depending on mechanotransduction upon the formation of PD-1/monoclonal antibody (mAb)/Fc-gamma receptor (Fc-γR) axes resulting from the interaction between PD-1/mAb complex, on the surface of tumor-specific T-cells, and Fc-γR-bearing tumor-associated macrophages (TAMs) within the tumor microenvironment. The generated mechanical force activates ITIM and ITSM on the cytoplasmic endodomain of the PD-1 receptor, leading to suppression of the effector function of tumor-specific T-cells which effectively unleashes cancer cells from the cytotoxic barrier and causes HPD in affected patients. This model provides clues about why patients receiving anti-PD1/PD-L1 mAbs are more prone to develop HPD as well as the variability of the ICIs response among treated patients. Additionally, it features the effect of specific immunophenotypic dynamics, such as TAM infiltration, on the final outcome of antibody-based immunotherapy and gives new insights for designing next-generation immunomodulatory interventions.

Role of Mechanotransduction and Tension in T Cell Function.

T cell migration from blood to, and within lymphoid organs and tissue, as well as, T cell activation rely on complex biochemical signaling events. But T cell migration and activation also take place in distinct mechanical environments and lead to drastic morphological changes and reorganization of the acto-myosin cytoskeleton. In this review we discuss how adhesion proteins and the T cell receptor act as mechanosensors to translate these mechanical contexts into signaling events. We further discuss how cell tension could bring a significant contribution to the regulation of T cell signaling and function.

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques.

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the Förster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Piezo proteins: incidence and abundance in the enteric nervous system. Is there a link with mechanosensitivity?

Piezo channels play fundamental roles in many physiological processes. Their presence and functional role in the enteric nervous system is still not known. We hypothesize that they play a role in mechanotransduction in enteric neurons. Our aims are to quantify the presence of both Piezo1 and 2 in enteric neurons throughout the gastrointestinal tract using immunohistochemistry and analyze their function(s) using neuroimaging techniques and pharmacological investigations. In order to perform a systematic and comparative study, we performed our experiments in gastrointestinal tissue from guinea pigs, mice and humans. Piezo1 (20-70%) is expressed by both enteric neuronal cell bodies and fibers in the myenteric and submucosal plexi of all the species investigated. Generally, Piezo1 expressing somata are more numerous in the submucosal plexus (50-80%) than in the myenteric plexus (15-35%) apart from the stomach where Piezo1 is expressed in up to 60% of cell bodies. Myenteric Piezo1 neurons mainly (60-100%) but not exclusively, also express nitric oxide synthase, a minority express choline acetyltransferase. In the submucosal plexus, Piezo1 neurons co-express vasoactive intestinal peptide (40-90%). Conversely, expression of Piezo2 is extremely rare in the somata of enteric neurons and is present in few neurites. In functional experiments, 38-76% of the mechanosensitive neurons expressed Piezo1 channels. Statistical analysis showed a positive significant correlation between mechanosensitive and Piezo1 positive neurons. However, pharmacological experiments using an activator and an inhibitor of Piezo channels did not demonstrate changes in mechanotransduction. A major role of Piezo1 in the mechanosensitivity of enteric neurons can be excluded.

Functional regeneration of tendons using scaffolds with physical anisotropy engineered via microarchitectural manipulation.

Structural and hierarchical anisotropy underlies the structure-function relationship of most living tissues. Attempts to exploit the interplay between cells and their immediate environment have rarely featured macroscale, three-dimensional constructs required for clinical applications. Furthermore, compromises to biomechanical robustness during fabrication often limit the scaffold's relevance in translational medicine. We report a polymeric three-dimensional scaffold with tendon-like mechanical properties and controlled anisotropic microstructures. The scaffold was composed of two distinct portions, which enabled high porosity while retaining tendon-like mechanical properties. When tenocytes were cultured in vitro on the scaffold, phenotypic markers of tenogenesis such as type-I collagen, decorin, and tenascin were significantly expressed over nonanisotropic controls. Moreover, highly aligned intracellular cytoskeletal network and high nuclear alignment efficiencies were observed, suggesting that microstructural anisotropy might play the epigenetic role of mechanotransduction. When implanted in an in vivo micropig model, a neotissue that formed over the scaffold resembled native tendon tissue in composition and structure.

Can We Improve Vaginal Tissue Healing Using Customized Devices: 3D Printing and Biomechanical Changes in Vaginal Tissue

Background: Determining biomechanical changes in vaginal tissue with tissue stretch is critical for understanding the role of mechanotransduction on vaginal tissue healing. Noncontact dynamic optical coherence elastography (OCE) can quantify biomechanical changes in vaginal tissues noninvasively. Improved vaginal tissue healing will reduce postoperative complications from vaginal surgery. Aims: (1) To complete dimensional assessments (DAs) of the vaginal tract. (2) To elucidate biomechanical properties (BMP) of porcine vaginal tissues (PVT). (3) Compare BMPs of piglet and adult PVTs after placement of customized vaginal dilators (VD) by OCE and uniaxial mechanical testing (MT). Methods: Pilot study using adult nulliparous pig and piglet PVTs (n = 20 each). DA of PVTs was performed using silicone molding. 3D-printed VDs were used to achieve different Relative Diameter Change (RDC) of the PVTs (no dilatation, and -50%, 0%, 50% RDC). Elastographic testing using OCE and MT. Results: Using OCE, no significant differences (SD) were noted between adult and piglet PVT (p = 0.74) or by stretch direction (p = 0.300). SD was noted with increasing RDC (p = 0.023). Using MT, there were SD in tissue stiffness between adult and piglet PVT (p = 0.048), but no SD as a function of RDC (p = 0.750) or stretch direction (p = 0.592). Conclusions: This study quantified biomechanical changes in PVT with customized stretching by 3D printed VD using both OCE and MT. This work has implications for the mechanotransduction of vaginal wound healing and noninvasive assessment of vaginal diseases.

The roles of mechanotransduction, vascular wall cells, and blood cells in atheroma induction.

This paper describes and analyzes the cellular and molecular mechanisms underlying atherosclerosis development. In particular, the roles of monocytes/macrophages, smooth muscle cells, and vascular endothelium in the formation of stable/unstable atheromatous plaques, and the contributions of some processes to atheroma formation. In this study we analyzed endothelium: function, dysfunction, and involvement into atherogenesis; cell proteins mediating mechanotransduction; proatherogenic role of monocytes; the role of macrophages in the development of unstable atheromatous plaques and smooth muscle cell origin in atherosclerosis. Smooth muscle cell phenotypic switching; their functioning; the ability to retain cholesterol and lipoproteins as well as secretion of pro- and anti-inflammatory molecules and extracellular matrix proteins, their response to extracellular stimuli secreted by other cells, and the effect of smooth muscle cells on the cells surrounding atheromatous plaques are fundamentally important for the insight into atherosclerosis molecular basis. Atheromatous plaque transcriptome studies will be helpful in the identification of the key genes involved in atheroma transformation and development as well as discovery of the new targets for diagnosis and therapy.

Effects of low-magnitude high-frequency vibration on osteoblasts are dependent on estrogen receptor α signaling and cytoskeletal remodeling

Clinical and experimental studies demonstrate the potential of low-magnitude high-frequency vibration (LMHFV) to enhance bone formation in the intact skeleton and during fracture healing. Moreover, it was shown that the effects of vibration therapy during fracture healing are highly dependent on the estrogen status of the vibrated individual and that estrogen receptor (ER) α signaling plays a major role in mechanotransduction of LMHFV. Because it is known that LMHFV can directly act on osteogenic cells, we hypothesize that the differential effects of LMHFV in the presence and absence of estrogen are mediated by ERα signaling in osteoblasts. To prove this hypothesis, we subjected preosteoblastic MC3T3-E1 cells and primary osteoblasts to LMHFV in vitro. We found increased Cox2 gene expression, cell metabolic activity and cell proliferation after LMHFV in the absence of estrogen, whereas the effects were contrary in the presence of estrogen. Blocking of ERα signaling by Esr1-siRNA knockdown or adding the selective ERα antagonist MPP dihydrochloride abolished the effects of LMHFV on osteoblast proliferation and Cox2 expression. Furthermore, primary osteoblasts isolated from ERα-knockout mice did not show a response towards LMHFV in the presence of estrogen. Additionally, blocking of actin cytoskeletal remodeling by adding the p160ROCK inhibitor Y-27632 abolished the effects of LMHFV. In contrast, expression of primary cilium was not necessary for mechanotransduction of LMHFV. These results suggest that direct effects of LMHFV on osteoblasts are dependent on ERα signaling and cytoskeletal remodeling.

Piezo1 channels are mechanosensors in human fetoplacental endothelial cells

STUDY QUESTIONDoes the shear stress sensing ion channel subunit Piezo1 have an important mechanotransduction role in human fetoplacental endothelium?SUMMARY ANSWERPiezo1 is present and functionally active in human fetoplacental endothelial cells, and disruption of Piezo1 prevents the normal response to shear stress.WHAT IS KNOWN ALREADYShear stress is an important stimulus for maturation and function of placental vasculature but the molecular mechanisms by which the force is detected and transduced are unclear. Piezo1 channels are Ca2+-permeable non-selective cationic channels which are critical for shear stress sensing and maturation of murine embryonic vasculature.STUDY DESIGN, SAMPLES/MATERIALS, METHODSWe investigated the relevance of Piezo1 to placental vasculature by studying human fetoplacental endothelial cells (FpECs) from healthy pregnancies. Endothelial cells were isolated from placental cotyledons and cultured, for the study of tube formation and cell alignment to shear stress. In addition, human placental arterial endothelial cells were isolated and studied immediately by patch-clamp electrophysiology.MAIN RESULTS AND THE ROLE OF CHANCEThe synthetic Piezo1 channel agonist Yoda1 caused strong elevation of the intracellular Ca2+ concentration with a 50% effect occurring at about 5.4 μM. Knockdown of Piezo1 by RNA interference suppressed the Yoda1 response, consistent with it being mediated by Piezo1 channels. Alignment of cells to the direction of shear stress was also suppressed by Piezo1 knockdown without loss of cell viability. Patch-clamp recordings from freshly isolated endothelium showed shear stress-activated single channels which were characteristic of Piezo1.LIMITATIONS, REASONS FOR CAUTIONThe in vitro nature of fetoplacental endothelial cell isolation and subsequent culture may affect FpEC characteristics and PIEZO1 expression. In addition to Piezo1, alternative shear stress sensing mechanisms have been suggested in other systems and might also contribute in the placenta.WIDER IMPLICATIONS OF THE FINDINGSThese data suggest that Piezo1 is an important molecular determinant of blood flow sensitivity in the placenta. Establishing and manipulating the molecular mechanisms regulating shear stress sensing could lead to novel therapeutic strategies to improve blood flow in the placenta.LARGE-SCALE DATANot applicable.STUDY FUNDING/COMPETING INTEREST(S)LCM was funded by a Clinical Research Training Fellowship from the Medical Research Council and by the Royal College of Obstetricians and Gynaecologists, and has received support from a Wellcome Trust Institutional Strategic Support Fund. JS was supported by the Wellcome Trust and a BHF Intermediate Research Fellowship. HJG, CW, AJH and PJW were supported by PhD Studentships from BHF, BBSRC and the Leeds Teaching Hospitals Charitable Foundation respectively. All authors declare no conflict of interest.

Mechanotransduction by PCDH15 Relies on a Novel cis-Dimeric Architecture

The tip link, a filament formed by protocadherin 15 (PCDH15) and cadherin 23, conveys mechanical force from sound waves and head movement toopen hair-cell mechanotransduction channels. Tip-link cadherins are thought to have acquired structural features critical for their role in mechanotransduction. Here, we biophysically and structurally characterize the unusual cis-homodimeric architecture of PCDH15. We show that PCDH15 molecules form double-helical assemblies through cis-dimerization interfaces in the extracellular cadherin EC2-EC3 domain region and in a unique membrane-proximal domain. Electron microscopy studies visualize the cis-dimeric PCDH15 assembly and reveal the PCDH15 extracellular domain as a parallel double helix with cis cross-bridges at the two locations we defined. Thehelical configuration suggests the potential forelasticity through helix winding and unwinding. Functional studies in hair cells show that mutations that perturb PCDH15 dimerization contacts affect mechanotransduction. Together, these data reveal the cis-dimeric architecture of PCDH15 and show that dimerization is critical for sensing mechanical stimuli.

Molecular mechanisms of mechanotransduction in psoriasis.

Psoriasis is an immune disease of the skin that frequently develops upon triggering events of mechanical nature and leads to increased proliferation and damaged differentiation of keratinocytes of the epidermis. Mechanical forces are mediated through mechanotransduction, which is the process that translates physical cues into biochemical signaling networks. Latest updates underline the role of mechanotransduction during the acquisition of aberrant properties by the keratinocytes of the skin, therefore implying a potential contribution that promotes psoriasis pathogenesis. The present review discusses the mechano-induced signaling pathways and individual molecules that become activated in psoriasis and in keratinocytes, along with mechano-based putative treatment strategies. We also suggest emerging mechanosensitive molecules for further investigation with potential diagnostic and therapeutic utility in psoriasis.

β-Catenin–dependent mechanotransduction dates back to the common ancestor of Cnidaria and Bilateria

Although the genetic regulation of cellular differentiation processes is well established, recent studies have revealed the role of mechanotransduction on a variety of biological processes, including regulation of gene expression. However, it remains unclear how universal and widespread mechanotransduction is in embryonic development of animals. Here, we investigate mechanosensitive gene expression during gastrulation of the starlet sea anemone Nematostella vectensis, a cnidarian model organism. We show that the blastoporal marker gene brachyury is down-regulated by blocking myosin II-dependent gastrulation movements. Brachyury expression can be restored by applying external mechanical force. Using CRISPR/Cas9 and morpholino antisense technology, we also show that mechanotransduction leading to brachyury expression is β-catenin dependent, similar to recent findings in fish and Drosophila [Brunet T, et al. (2013) Nat Commun 4:1-15]. Finally, we demonstrate that prolonged application of mechanical stress on the embryo leads to ectopic brachyury expression. Thus, our data indicate that β-catenin-dependent mechanotransduction is an ancient gene regulatory mechanism, which was present in the common ancestor of cnidarians and bilaterians, at least 600 million years ago.

Vascular Cell Glycocalyx-Mediated Vascular Remodeling Induced by Hemodynamic Environmental Alteration.

Vascular remodeling induced by hemodynamic stimuli contributes to the pathophysiology of cardiovascular diseases. The importance of vascular cells (endothelial cells and smooth muscle cells) glycocalyx in the mechanotransduction of flow-induced shear stress at the cellular and molecular levels has been demonstrated over the past decade. However, its potential mechanotransduction role in vascular remodeling has triggered little attention. In the present study, a home-made apparatus was used to expose the rat abdominal aorta to sterile, flow or no flow, normal-pressure or high-pressure conditions for 4 days. The histomophometric, cellular, and molecular analysis of vessels were performed. The results showed that after exposing the vessels in the flow and high-pressure condition, the apoptotic rate, the cell number, and the RNA level of contractile marker gene smooth muscle 22 of smooth muscle cells were significantly increased, whereas the expression of nitric oxide synthase, α-smooth muscle actin, smoothelin, and calponion showed no significant differences compared with the flow and normal-pressure groups. Moreover, the histomophometric analysis of vascular walls suggested a remodeling induced by flow and high-pressure loading consistent with the classic hypertensive aortic phenotype, which is characterized by a thicker and more rigid vascular wall as well as increased aortic diameter. However, those phenomena were totally abolished after compromising the integrity of glycocalyx by the treatment of vessels with hyaluronidase, which provided evidence of the important mechanotransduction role of the vascular cells glycocalyx in vascular remodeling induced by hemodynamic stimuli.

Actin retrograde flow controls natural killer cell response by regulating the conformation state of SHP-1.

Natural killer (NK) cells are a powerful weapon against viral infections and tumor growth. Although the actin-myosin (actomyosin) cytoskeleton is crucial for a variety of cellular processes, the role of mechanotransduction, the conversion of actomyosin mechanical forces into signaling cascades, was never explored in NK cells. Here, we demonstrate that actomyosin retrograde flow (ARF) controls the immune response of primary human NK cells through a novel interaction between β-actin and the SH2-domain-containing protein tyrosine phosphatase-1 (SHP-1), converting its conformation state, and thereby regulating NK cell cytotoxicity. Our results identify ARF as a master regulator of the NK cell immune response. Since actin dynamics occur in multiple cellular processes, this mechanism might also regulate the activity of SHP-1 in additional cellular systems.

Characterization of bone morphology in CCN5/WISP5 knockout mice.

CCN5/WISP2 is part of the CCN family of matricellular proteins, but is distinct in that it lacks the C-terminal (CT) domain. Although CCN5 has been shown to impact cell proliferation and differentiation in vitro, its role in vivo is unclear. We therefore generated mice using ES cells developed by the Knockout Mouse Project (KOMP) in which exons 2-5, which encode the all of the conserved protein coding regions, are replaced by a lacZ cassette. Ccn5 LacZ/LacZ mice were viable and apparently normal. Based on previous studies showing that CCN5 impacts osteoblast proliferation and differentiation, we performed an analysis of adult bone phenotype. LacZ expression was examined in adult bone, and was found to be strong within the periosteum, but not in trabecular bone or bone marrow. Micro-CT analysis revealed no apparent changes in bone mineral density (BMD) or bone tissue volume (BV/TV) in Ccn5 LacZ/LacZ mice. These studies indicate that CCN5 is not required for normal bone formation, but they do not rule out a role in mechanotransduction or repair processes. The availability of Ccn5 LacZ mice enables studies of CCN5 expression and function in multiple tissues.