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

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

Self-organized insulin-producing ß-cells differentiated from human omentum-derived stem cells and their in vivo therapeutic potential.

BACKGROUND: Human omentum-derived mesenchymal stem cells (hO-MSCs) possess great potential to differentiate into multiple lineages and have self-renewal capacity, allowing them to be utilized as patient-specific cell-based therapeutics. Although the use of various stem cell-derived ß-cells has been proposed as a novel approach for treating diabetes mellitus, developing an efficient method to establish highly functional ß-cells remains challenging. METHODS: We aimed to develop a novel cell culture platform that utilizes a fibroblast growth factor 2 (FGF2)-immobilized matrix to regulate the adhesion and differentiation of hO-MSCs into insulin-producing ß-cells via cell-matrix/cell-cell interactions. In our study, we evaluated the in vitro differentiation potential of hO-MSCs cultured on an FGF2-immobilized matrix and a round-bottom plate (RBP). Further, the in vivo therapeutic efficacy of the ß-cells transplanted into kidney capsules was evaluated using animal models with streptozotocin (STZ)-induced diabetes. RESULTS: Our findings demonstrated that cells cultured on an FGF2-immobilized matrix could self-organize into insulin-producing ß-cell progenitors, as evident from the upregulation of pancreatic ß-cell-specific markers (PDX-1, Insulin, and Glut-2). Moreover, we observed significant upregulation of heparan sulfate proteoglycan, gap junction proteins (Cx36 and Cx43), and cell adhesion molecules (E-cadherin and Ncam1) in cells cultured on the FGF2-immobilized matrix. In addition, in vivo transplantation of differentiated ß-cells into animal models of STZ-induced diabetes revealed their survival and engraftment as well as glucose-sensitive production of insulin within the host microenvironment, at over 4 weeks after transplantation. CONCLUSIONS: Our findings suggest that the FGF2-immobilized matrix can support initial cell adhesion, maturation, and glucose-stimulated insulin secretion within the host microenvironment. Such a cell culture platform can offer novel strategies to obtain functional pancreatic ß-cells from patient-specific cell sources, ultimately enabling better treatment for diabetes mellitus.

Heparin-mimicking polymer-based hydrogel matrix regulates macrophage polarization by controlling cell adhesion.

The physicochemical properties of biomaterials influence cell adhesion, shape, and polarization of macrophages. In this study, we aimed to evaluate the polarization of macrophages in terms of the regulation of cell adhesion and how synthetic mimics for heparin and poly(sodium-4-styrenesulfonate) can regulate macrophage polarization by modulating cell shape, focal adhesion, cell traction force, and intracellular tension. Our initial findings showed that macrophages cultured with heparin-mimicking polymer-based hydrogel matrix showed reduced expression of cell adhesion markers such as integrins, vinculin, RhoA, and ROCK1/2 and reduced cell shape, elongation, cell-matrix traction force, and intracellular tension. Furthermore, we observed a significant decrease in cell adhesion in cells cultured on the hydrogel, resulting in the promotion of M1 polarization. These findings offer insights into the important roles of cell-matrix interactions in macrophage polarization and offer a platform for heparin-mimicking polymer-based hydrogel matrices to induce M1 polarization by inducing cell adhesion without classical activators.

A Novel Strategy for Creating an Antibacterial Surface Using a Highly Efficient Electrospray-Based Method for Silica Deposition.

Adhesion of bacteria on biomedical implant surfaces is a prerequisite for biofilm formation, which may increase the chances of infection and chronic inflammation. In this study, we employed a novel electrospray-based technique to develop an antibacterial surface by efficiently depositing silica homogeneously onto polyethylene terephthalate (PET) film to achieve hydrophobic and anti-adhesive properties. We evaluated its potential application in inhibiting bacterial adhesion using both Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) bacteria. These silica-deposited PET surfaces could provide hydrophobic surfaces with a water contact angle greater than 120° as well as increased surface roughness (root mean square roughness value of 82.50 ± 16.22 nm and average roughness value of 65.15 ± 15.26 nm) that could significantly reduce bacterial adhesion by approximately 66.30% and 64.09% for E. coli and S. aureus, respectively, compared with those on plain PET surfaces. Furthermore, we observed that silica-deposited PET surfaces showed no detrimental effects on cell viability in human dermal fibroblasts, as confirmed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide and live/dead assays. Taken together, such approaches that are easy to synthesize, cost effective, and efficient, and could provide innovative strategies for preventing bacterial adhesion on biomedical implant surfaces in the clinical setting.

Heparin-Mimicking Polymer-Based In Vitro Platform Recapitulates In Vivo Muscle Atrophy Phenotypes.

The cell-cell/cell-matrix interactions between myoblasts and their extracellular microenvironment have been shown to play a crucial role in the regulation of in vitro myogenic differentiation and in vivo skeletal muscle regeneration. In this study, by harnessing the heparin-mimicking polymer, poly(sodium-4-styrenesulfonate) (PSS), which has a negatively charged surface, we engineered an in vitro cell culture platform for the purpose of recapitulating in vivo muscle atrophy-like phenotypes. Our initial findings showed that heparin-mimicking moieties inhibited the fusion of mononucleated myoblasts into multinucleated myotubes, as indicated by the decreased gene and protein expression levels of myogenic factors, myotube fusion-related markers, and focal adhesion kinase (FAK). We further elucidated the underlying molecular mechanism via transcriptome analyses, observing that the insulin/PI3K/mTOR and Wnt signaling pathways were significantly downregulated by heparin-mimicking moieties through the inhibition of FAK/Cav3. Taken together, the easy-to-adapt heparin-mimicking polymer-based in vitro cell culture platform could be an attractive platform for potential applications in drug screening, providing clear readouts of changes in insulin/PI3K/mTOR and Wnt signaling pathways.

Ameliorating Fibrotic Phenotypes of Keloid Dermal Fibroblasts through an Epidermal Growth Factor-Mediated Extracellular Matrix Remodeling.

Keloid and hypertrophic scars are skin fibrosis-associated disorders that exhibit an uncontrollable proliferation of fibroblasts and their subsequent contribution to the excessive accumulation of extracellular matrix (ECM) in the dermis. In this study, to elucidate the underlying mechanisms, we investigated the pivotal roles of epidermal growth factor (EGF) in modulating fibrotic phenotypes of keloid and hypertrophic dermal fibroblasts. Our initial findings revealed the molecular signatures of keloid dermal fibroblasts and showed the highest degree of skin fibrosis markers, ECM remodeling, anabolic collagen-cross-linking enzymes, such as lysyl oxidase (LOX) and four LOX-like family enzymes, migration ability, and cell-matrix traction force, at cell-matrix interfaces. Furthermore, we observed significant EGF-mediated downregulation of anabolic collagen-cross-linking enzymes, resulting in amelioration of fibrotic phenotypes and a decrease in cell motility measured according to the cell-matrix traction force. These findings offer insight into the important roles of EGF-mediated cell-matrix interactions at the cell-matrix interface, as well as ECM remodeling. Furthermore, the results suggest their contribution to the reduction of fibrotic phenotypes in keloid dermal fibroblasts, which could lead to the development of therapeutic modalities to prevent or reduce scar tissue formation.

Traction force microscopy for understanding cellular mechanotransduction.

Under physiological and pathological conditions, mechanical forces generated from cells themselves or transmitted from extracellular matrix (ECM) through focal adhesions (FAs) and adherens junctions (AJs) are known to play a significant role in regulating various cell behaviors. Substantial progresses have been made in the field of mechanobiology towards novel methods to understand how cells are able to sense and adapt to these mechanical forces over the years. To address these issues, this review will discuss recent advancements of traction force microscopy (TFM), intracellular force microscopy (IFM), and monolayer stress microscopy (MSM) to measure multiple aspects of cellular forces exerted by cells at cell-ECM and cell-cell junctional intracellular interfaces. We will also highlight how these methods can elucidate the roles of mechanical forces at interfaces of cell-cell/cell-ECM in regulating various cellular functions. [BMB Reports 2020; 53(2): 74-81].

3D Printed Templating of Extrinsic Freeze-Casting for Macro-Microporous Biomaterials.

As with most biological materials, natural bone has hierarchical structure. The microstructural features of compact bone are of various length scales with its porosity consisting of larger osteons (∼100 µm diameter) and vascular channels, as well as smaller lacuna spaces (∼10 µm diameter). In this study, the freeze-casting process, which has been previously used to form biocompatible porous scaffolds (made with hydroxyapatite, HA) has been improved to mimic the intrinsic hierarchical structure of natural bone by implementing an extrinsic 3D printed template. The results of pore characterization showed that this novel combined method of 3D printing and freeze-casting is able to produce porosity at multiple length scales. Nonporous, microporous (created with freeze-casting alone), and macro-micro porous (created with freeze-casting and 3D printed templating) scaffolds were compared as substrates to evaluate cellular activities using osteoblast-like MG63 cell lines. The number of cells oriented parallel to the HA wall structures in the freeze-cast scaffold was found to increase on the microporous and macro-micro porous samples when compare to the nonporous samples, mimicking the natural alignment of the lamella of natural bone. Regarding the cell morphologies, cells on microporous and macro-micro porous samples showed narrowly aligned shapes, whereas those on nonporous samples had polygonal shapes with no discernible orientation. Proliferation and differentiation tests demonstrated that no toxicity or functional abnormalities were found in any of the substrates due to potential chemical and mechanical residues that may have been introduced by the freeze-casting process. Monitoring of the three-dimensional distribution of cells in the scaffolds through microcomputed tomography indicates that the cells were well distributed in the interior pore spaces via the interpenetrating macro-micro pore networks. In summary, we demonstrate this novel approach can create porosity at multiple length scales and is highly favorable in creating a biocompatible, osteoconductive, and structurally hierarchical HA scaffolds for biomedical applications.

Lis1 dysfunction leads to traction force reduction and cytoskeletal disorganization during cell migration.

Cell migration is a critical process during development, tissue repair, and cancer metastasis. It requires complex processes of cell adhesion, cytoskeletal dynamics, and force generation. Lis1 plays an important role in the migration of neurons, fibroblasts and other cell types, and is essential for normal development of the cerebral cortex. Mutations in human LIS1 gene cause classical lissencephaly (smooth brain), resulting from defects in neuronal migration. However, how Lis1 may affect force generation in migrating cells is still not fully understood. Using traction force microscopy (TFM) with live cell imaging to measure cellular traction force in migrating NIH3T3 cells, we showed that Lis1 knockdown (KD) by RNA interference (RNAi) caused reductions in cell migration and traction force against the extracellular matrix (ECM). Immunostaining of cytoskeletal components in Lis1 KD cells showed disorganization of microtubules and actin filaments. Interestingly, focal adhesions at the cell periphery were significantly reduced. These results suggest that Lis1 is important for cellular traction force generation through the regulation of cytoskeleton organization and focal adhesion formation in migrating cells.

Matrix stiffness determines the phenotype of vascular smooth muscle cell in vitro and in vivo: Role of DNA methyltransferase 1.

Cells perceive the physical cues such as perturbations of extracellular matrix (ECM) stiffness, and translate these stimuli into biochemical signals controlling various aspects of cell behavior, which contribute to the physiological and pathological processes of multiple organs. In this study, we tested the hypothesis that during arterial stiffening, vascular smooth muscle cells (SMCs) sense the increase of ECM stiffness, which modulates the cellular phenotype through the regulation in DNA methyltransferases 1 (DNMT1) expression. Moreover, we hypothesized that the mechanisms involve intrinsic stiffening and deficiency in contractility of vascular SMCs. Substrate stiffening was mimicked in vitro with polyacrylamide gels. A contractile-to-synthetic phenotypic transition was induced by substrate stiffening in vascular SMCs through the down-regulation of DNMT1 expression. DNMT1 repression was also observed in the tunica media of mice aortas in an acute aortic injury model and a chronic kidney failure model, as well as in the tunica intima of human carotid arteries with calcified atherosclerotic lesions. DNMT1 inhibition facilitates arterial stiffening in vivo and promotes osteogenic transdifferentiation, calcification and cellular stiffening of vascular SMCs in vitro. These effects may be attributable, at least in part, to the role of DNMT1 in regulating the promoter activities of Transgelin (SM22α) and α-smooth muscle actin (SMA) and the functional contractility of SMCs. We conclude that DNMT1 is a critical regulator that negatively regulates arterial stiffening via maintaining the contractile phenotype of vascular SMCs. This research may facilitate elucidation of the complex crosstalk between vascular SMCs and their surrounding matrix in healthy and in pathological conditions and provide new insights into the implications for potential targeting of the phenotypic regulatory mechanisms in material-related therapeutic applications.

Dexamethasone-induced cellular tension requires a SGK1-stimulated Sec5-GEF-H1 interaction.

Dexamethasone, a synthetic glucocorticoid, is often used to induce osteoblast commitment of mesenchymal stem cells (MSCs), and this process requires RhoA-dependent cellular tension. The underlying mechanism is unclear. In this study, we show that dexamethasone stimulates expression of fibronectin and integrin α5 (ITGA5), accompanied by an increase in the interaction of GEF-H1 (also known as ARHGEF2) with Sec5 (also known as EXOC2), a microtubule (MT)-regulated RhoA activator and a component of the exocyst, respectively. Disruption of this interaction abolishes dexamethasone-induced cellular tension and GEF-H1 targeting to focal adhesion sites at the cell periphery without affecting dexamethasone-induced levels of ITGA5 and fibronectin, and the extracellular deposition of fibronectin at adhesion sites is specifically inhibited. We demonstrate that dexamethasone stimulates the expression of serum-glucocorticoid-induced protein kinase 1 (SGK1), which is necessary and sufficient for the induction of the Sec5-GEF-H1 interaction. Given the function of SGK1 in suppressing MT growth, our data suggest that the induction of SGK1 through treatment with dexamethasone alters MT dynamics to increase Sec5-GEF-H1 interactions, which promote GEF-H1 targeting to adhesion sites. This mechanism is essential for the formation of fibronectin fibrils and their attachment to integrins at adhesion sites in order to generate cellular tension.

Piezo1, a mechanically activated ion channel, is required for vascular development in mice.

Mechanosensation is perhaps the last sensory modality not understood at the molecular level. Ion channels that sense mechanical force are postulated to play critical roles in a variety of biological processes including sensing touch/pain (somatosensation), sound (hearing), and shear stress (cardiovascular physiology); however, the identity of these ion channels has remained elusive. We previously identified Piezo1 and Piezo2 as mechanically activated cation channels that are expressed in many mechanosensitive cell types. Here, we show that Piezo1 is expressed in endothelial cells of developing blood vessels in mice. Piezo1-deficient embryos die at midgestation with defects in vascular remodeling, a process critically influenced by blood flow. We demonstrate that Piezo1 is activated by shear stress, the major type of mechanical force experienced by endothelial cells in response to blood flow. Furthermore, loss of Piezo1 in endothelial cells leads to deficits in stress fiber and cellular orientation in response to shear stress, linking Piezo1 mechanotransduction to regulation of cell morphology. These findings highlight an essential role of mammalian Piezo1 in vascular development during embryonic development.

Shp2 plays a crucial role in cell structural orientation and force polarity in response to matrix rigidity.

Cells can sense and respond to physical properties of their surrounding extracellular matrix. We have demonstrated here that tyrosine phosphatase Shp2 plays an essential role in the response of mouse embryonic fibroblasts to matrix rigidity. On rigid surfaces, large focal adhesions (FAs) and anisotropically oriented stress fibers are formed, whereas cells plated on compliant substrates form numerous small FAs and radially oriented stress fibers. As a result, traction force is increased and organized to promote cell spreading and elongation on rigid substrates. Shp2-deficient cells do not exhibit the stiffness-dependent increase in FA size and polarized stress fibers nor the intracellular tension and cell shape change. These results indicate the involvement of Shp2 in regulating the FAs and the cytoskeleton for force maintenance and organization. The defect of FA maturation in Shp2-deficient cells was rescued by expressing Y722F Rho-associated protein kinase II (ROCKII), suggesting that ROCKII is the molecular target of Shp2 in FAs for the FA maturation. Thus, Shp2 serves as a key mediator in FAs for the regulation of structural organization and force orientation of mouse embyonic fibroblasts in determining their mechanical polarity in response to matrix rigidity.

Matrix stiffness regulates endothelial cell proliferation through septin 9.

Endothelial proliferation, which is an important process in vascular homeostasis, can be regulated by the extracellular microenvironment. In this study we demonstrated that proliferation of endothelial cells (ECs) was enhanced on hydrogels with high stiffness (HSG, 21.5 kPa) in comparison to those with low stiffness (LSG, 1.72 kPa). ECs on HSG showed markedly prominent stress fibers and a higher RhoA activity than ECs on LSG. Blockade of RhoA attenuated stress fiber formation and proliferation of ECs on HSG, but had little effect on ECs on LSG; enhancement of RhoA had opposite effects. The phosphorylations of Src and Vav2, which are positive RhoA upstream effectors, were higher in ECs on HSG. The inhibition of Src/Vav2 attenuated the HSG-mediated RhoA activation and EC proliferation but exhibited nominal effects on ECs on LSG. Septin 9 (SEPT9), the negative upstream effector for RhoA, was significantly higher in ECs on LSG. The inhibition of SEPT9 increased RhoA activation, Src/Vav2 phosphorylations, and EC proliferation on LSG, but showed minor effects on ECs on HSG. We further demonstrated that the inactivation of integrin α(v)ß(3) caused an increase of SEPT9 expression in ECs on HSG to attenuate Src/Vav2 phosphorylations and inhibit RhoA-dependent EC proliferation. These results demonstrate that the SEPT9/Src/Vav2/RhoA pathway constitutes an important molecular mechanism for the mechanical regulation of EC proliferation.

Roles of cell confluency and fluid shear in 3-dimensional intracellular forces in endothelial cells.

We use a novel 3D inter-/intracellular force microscopy technique based on 3D traction force microscopy to measure the cell-cell junctional and intracellular tensions in subconfluent and confluent vascular endothelial cell (EC) monolayers under static and shear flow conditions. We found that z-direction cell-cell junctional tensions are higher in confluent EC monolayers than those in subconfluent ECs, which cannot be revealed in the previous 2D methods. Under static conditions, subconfluent cells are under spatially non-uniform tensions, whereas cells in confluent monolayers are under uniform tensions. The shear modulations of EC cytoskeletal remodeling, extracellular matrix (ECM) adhesions, and cell-cell junctions lead to significant changes in intracellular tensions. When a confluent monolayer is subjected to flow shear stresses with a high forward component comparable to that seen in the straight part of the arterial system, the intracellular and junction tensions preferentially increase along the flow direction over time, which may be related to the relocation of adherens junction proteins. The increases in intracellular tensions are shown to be a result of chemo-mechanical responses of the ECs under flow shear rather than a direct result of mechanical loading. In contrast, the intracellular tensions do not show a preferential orientation under oscillatory flow with a very low mean shear. These differences in the directionality and magnitude of intracellular tensions may modulate translation and transcription of ECs under different flow patterns, thus affecting their susceptibility for atherogenesis.

Live Cells Exert 3-Dimensional Traction Forces on Their Substrata.

The traction forces exerted by an adherent cell on a substrate have been studied only in the two-dimensions (2D) tangential to substrate surface (Txy). We developed a novel technique to measure the three-dimensional (3D) traction forces exerted by live bovine aortic endothelial cells (BAECs) on polyacrylamide deformable substrate. On 3D images acquired by confocal microscopy, displacements were determined with image-processing programs, and traction forces in tangential (XY) and normal (Z) directions were computed by finite element method (FEM). BAECs generated traction force in normal direction (Tz) with an order of magnitude comparable to Txy. Tz is upward at the cell edge and downward under the nucleus, changing continuously with a sign reversal between cell edge and nucleus edge. The method was evaluated regarding accuracy and precision of displacement measurements, effects of FE mesh size, displacement noises, and simple bootstrapping. These results provide new insights into cell-matrix interactions in terms of spatial and temporal variations in traction forces in 3D. This technique can be applied to study live cells to assess their biomechanical dynamics in conjunction with biochemical and functional activities, for investigating cellular functions in health and disease. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12195-009-0082-6) contains supplementary material, which is available to authorized users.