Inverse regulation of preproendothelin-1 and endothelin-converting enzyme-1beta genes in cardiac cells by mechanical load

Mechanical stretch and para- and/or autocrine factors, including endothelin-1, induce hypertrophy of cardiac myocytes and proliferation of fibroblasts. To investigate the effect of mechanical load on endothelin-1 production and endothelin system gene expression in neonatal rat ventricular myocytes and fibroblasts, we exposed cells to cyclic mechanical stretch in vitro (0.5 Hz, 10-25% elongation, from 1 min to 24 h). Endothelin-1 peptide levels were measured from culture media of myocytes and fibroblasts and human umbilical vein endothelial cells (positive control) by specific radioimmunoassay. Preproendothelin-1 promoter activity was measured via transfection of reporter plasmids and mRNA levels with Northern blot analysis or quantitative RT-PCR. Activity of extracellular signal-regulated kinase was quantified with specific kinase assay. We found that stretching of myocytes activated preproendothelin-1 gene expression, including promoter activation, transient mRNA level increases, and augmented endothelin-1 secretion. In contrast, preproendothelin-1 gene expression was inhibited in stretched fibroblasts. Endothelin-converting enzyme-1beta mRNA levels elevated in stretched fibroblasts but decreased in stretched myocytes. Endothelin receptor type A mRNA levels declined in stretched myocytes, whereas levels were below detection in fibroblasts. Stretch activated extracellular signal-regulated kinase in myocytes, and when the kinase activity was pharmacologically inhibited, the preproendothelin-1 induction was suppressed. Transient overexpression of mitogen-activated ERK-activating kinase-1 induced preproendothelin-1 promoter in myocytes. In summary, mechanical stretch distinctly regulates endothelin system gene expression in cardiac myocytes and fibroblasts. The inhibition of the endothelin system may affect cardiac mechanotransduction and therefore provides an approach in treatment of load-induced cardiac pathology.     

Mechanisms involved in regulation of osteoclastic differentiation by mechanical stress-loaded osteoblasts

Mechanical stress is known to be important for regulation of bone turnover, though the detailed mechanisms are not fully understood. In the present study, we examined the effect of mechanical stress on osteoblasts using a novel compression model. Mouse osteoblastic MC3T3-E1 cells were embedded in three-dimensional (3D) gels and cultured with continuous compressive force (0–10.0 g/cm²) for 48 h, and the conditioned medium were collected. RAW264.7 cells were then incubated with the conditioned medium for various times in the presence of receptor activator of nuclear factor-κB ligand (RANKL). Conditioned medium was found to inhibit the differentiation of RAW264.7 cells into osteoclasts induced by RANKL via down-regulation of the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), phosphorylation of IκBα, and nuclear translocation of p50 and p65. Interestingly, the conditioned medium also had a high level of binding activity to RANKL and blocked the binding of RANK to RANKL. Furthermore, the binding activity of conditioned medium to RANKL was reduced when the 3D gel was supplemented with KN-93, an inhibitor of non-canonical Wnt/Ca²⁺ pathway. In addition, expression level of osteoprotegerin (OPG) mRNA was increased in time- and force-dependent manners, and remarkably suppressed by KN-93. These results indicate that osteoblastic cells subjected to mechanical stress produce OPG, which binds to RANKL. Furthermore, this binding activity strongly inhibited osteoclastogenesis through suppression of TRAF6 and the nuclear factor-kappa B (NF-κB) signaling pathway, suggesting that enhancement of OPG expression induced by mechanical stress is dependent on non-canonical Wnt/Ca²⁺ pathway.     

Intercellular communication via gap junctions affected by mechanical load in the bovine annulus fibrosus

Cells in the intervertebral disc, as in other connective tissues including tendon, ligament and bone, form interconnected cellular networks that are linked via functional gap junctions. These cellular networks may be necessary to affect a coordinated response to mechanical and environmental stimuli. Using confocal microscopy with fluorescence recovery after photobleaching methods, we explored the in situ strain environment of the outer annulus of an intact bovine disc and the effect of high-level flexion on gap junction signalling. The in situ strain environment in the extracellular matrix of the outer annulus under high flexion load was observed to be non-uniform with the extensive cellular processes remaining crimped sometimes at flexion angles greater than 25°. A significant transient disruption of intercellular communication via functional gap junctions was measured after 10 and 20 min under high flexion load. This study illustrates that in healthy annulus fibrosus tissue, high mechanical loads can impede the functioning of the gap junctions. Future studies will explore more complex loading conditions to determine whether losses in intercellular communication can be permanent and whether gap junctions in aged and degenerated tissues become more susceptible to load. The current research suggests that cellular structures such as gap junctions and intercellular networks, as well as other cell–cell and cell–matrix interconnections, need to be considered in computational models in order to fully understand how macroscale mechanical signals are transmitted across scales to the microscale and ultimately into a cellular biosynthetic response in collagenous tissues.     

The layered structure of coronary adventitia under mechanical load

The mechanical loading-deformation relation of elastin and collagen fibril bundles is fundamental to understanding the microstructural properties of tissue. Here, we use multiphoton microscopy to obtain quantitative data of elastin and collagen fiber bundles under in situ loading of coronary adventitia. Simultaneous loading-imaging experiments on unstained fresh coronary adventitia allowed morphometric measurements of collagen and elastin fibril bundles and their individual deformation. Fiber data were analyzed at five different distension loading points (circumferential stretch ratio λ_θ = 1.0, 1.2, 1.4, 1.6, and 1.8) at a physiological axial stretch ratio of λ_axial = 1.3. Four fiber geometrical parameters were used to quantify the fibers: orientation angle, waviness, width, and area fraction. The results show that elastin and collagen fibers in inner adventitia form concentric densely packed fiber sheets, and the fiber orientation angle, width, and area fraction vary transmurally. The extent of fiber deformation depends on the initial orientation angle at no-distension state (λ_θ = 1.0 and λ_axial = 1.3). At higher distension loading, the orientation angle and waviness of fibers decrease linearly, but the width of collagen fiber is relatively constant at λ_θ = 1.0–1.4 and then decrease linearly for λ_θ ≥ 1.4. A decrease of the relative dispersion (SD/mean) of collagen fiber waviness suggests a heterogeneous mechanical response to loads. This study provides fundamental microstructural data for coronary artery biomechanics and we consider it seminal for structural models.     

Cell mechanical microenvironment for cell volume regulation

Cell volume regulation, as one of the fundamental homeostasis of the cell, is associated with many cellular behaviors and functions. With the increased studies on the effect of environmental mechanical cues on cell volume regulation, the relationship between cell volume regulation and mechanotransduction becomes more and more clear. In this paper, we review the mechanisms and hypotheses by which cell maintains its volume homeostasis both in vivo and in constructed cell mechanical microenvironment (CMM) in vitro. We discuss how the growth-division regulation maintains the volume homeostasis of cells in the cell cycle and how the cell cortex/membrane tension mediates the effect of CMM (i.e., osmotic pressure, matrix stiffness, and mechanical force) on cell volume regulation. We also highlight the roles of cell volume as a perfect integrator of the downstream signals of mechanotransduction from different aspects of CMM and an effective indicator for the mechanical condition that cell confronts. This interdisciplinary perspective can provide new insight into biomechanics and may shed light on bioengineering and pathological research work. We hope this review can facilitate future studies on the investigation of the role of cell volume in mechanotransduction.     

Chondrocyte suspension in fibrin glue

Autologous chondrocyte implantation has been shown to be a promising method for treatment of deep articular cartilage defects. The hyaline cartilage formed by implanted autologous chondrocytes has biomechanical properties similar to those of natural articular cartilage. Between June 2006 and September 2008 we performed Autologous chondrocyte implantation (ACI) in 50 patients and the chondrocytes were supported in fibrin glue. The cartilage biopsy samples were taken from the non-weight bearing area of the patient’s femoral condyle and the samples were transferred to the cell culture laboratory. Chondrocyte were kept in culture about 20 days. Fibrin glue was used as a three dimensional carrier for chondrocyte implantation. A 450 ml of patient’s own blood was collected prior to transplantation to produce autologous fibrinogen. Alternatively the allogenic fibrinogen was prepared from Regional Blood Center voluntary donors. Before surgery the chondrocyte suspension was mixed with fibrin glue and gel—like fibrograft was prepared. The total number of cells and the size of fibrograft depended on the defect size in the knee. Our results suggest that ACI technique with fibrin glue is a promising method for treatment of cartilage defect.     

Chondrocyte integrin expression and function

The extracellular matrix (ECM) is an "information rich" environment and interactions between the chondrocyte and ECM regulate many biological processes important to cartilage homeostasis and repair including cell attachment, growth, differentiation, and survival. The integrin family of cell surface receptors appears to play a major role in mediating cell-matrix interactions that are important in regulating these processes. Chondrocytes have been found to express several members of the integrin family which can serve as receptors for fibronectin (alpha 5 beta 1), types II and VI collagen (alpha 1 beta 1, alpha 2 beta 1, alpha 10 beta 1), laminin (alpha 6 beta 1), and vitronectin and osteopontin (alpha V beta 3). Integrin expression can be regulated by growth factors including IGF-I and TGF-beta. By providing a link between the ECM and the cytoskeleton, integrins may be important transducers of mechanical stimuli. Integrin binding stimulates intracellular signaling which can affect gene expression and regulate chondrocyte function. Further studies are needed to more clearly define the role of integrins in cartilage.     

Chondrocyte cell death mediated by reactive oxygen species-dependent activation of PKC-betaI

Signals generated by the extracellular matrix (ECM) promote cell survival. We have shown that chondrocytes detached from their native ECM and plated without serum at low density on poly-L-lysine undergo significant cell death that is associated with the production of reactive oxygen species (ROS). No cell death or ROS production was observed when cells were plated on fibronectin under the same conditions. Cell death on poly-L-lysine could be completely inhibited with the addition of either antioxidants or inhibitors of specific protein kinase C (PKC) isoforms including PKC-I. PKC-I was noted to translocate from the cytosol to the particulate membrane after plating on poly-L-lysine, and this translocation was inhibited by the addition of an antioxidant. Time-course analyses implicated endogenous ROS production as a secondary messenger leading to PKC-I activation and subsequent chondrocyte cell death. Cell survival on poly-L-lysine was significantly improved in the presence of oligomycin or DIDS, suggesting that ROS production occurred via complex V of the electron transport chain of the mitochondria and that ROS were released to the cytosol via voltage-dependent anion channels. Together, these results represent a novel mechanism by which ROS can initiate cell death through the activation of PKC-I. articular cartilage; osteoarthritis; cell signaling; fibronectin     

Contraction reaction: mechanical regulation of Rho GTPase

Epithelial cell differentiation is influenced by the physical environment. Local external changes in rigidity of the extracellular matrix are 'sensed' as increased or decreased tension and communicated intracellularly. The communications network that signals the state of the extracellular physical environment acts through the actin cytoskeleton to modulate Rho GTPase activity. A recent paper reports the surprising finding that breast epithelial cells respond to flexible surroundings by downregulating Rho activity through the actions of the Rho-regulated ROCK kinases.     

Redox regulation of diaphragm proteolysis during mechanical ventilation

Prevention of oxidative stress via antioxidants attenuates diaphragm myofiber atrophy associated with mechanical ventilation (MV). However, the specific redox-sensitive mechanisms responsible for this remain unknown. We tested the hypothesis that regulation of skeletal muscle proteolytic activity is a critical site of redox action during MV. Sprague-Dawley rats were assigned to five experimental groups: 1) control, 2) 6 h of MV, 3) 6 h of MV with infusion of the antioxidant Trolox, 4) 18 h of MV, and 5) 18 h of MV with Trolox. Trolox did not attenuate MV-induced increases in diaphragmatic levels of ubiquitin-protein conjugation, polyubiquitin mRNA, and gene expression of proteasomal subunits (20S proteasome alpha-subunit 7, 14-kDa E2, and proteasome-activating complex PA28). However, Trolox reduced both chymotrypsin-like and peptidylglutamyl peptide hydrolyzing (PGPH)-like 20S proteasome activities in the diaphragm after 18 h of MV. In addition, Trolox rescued diaphragm myofilament protein concentration (mug/mg muscle) and the percentage of easily releasable myofilament protein independent of alterations in ribosomal capacity for protein synthesis. In summary, these data are consistent with the notion that the protective effect of antioxidants on the diaphragm during MV is due, at least in part, to decreasing myofilament protein substrate availability to the proteasome.     

FimH forms catch bonds that are enhanced by mechanical force due to allosteric regulation

The bacterial adhesive protein, FimH, is the most common adhesin of Escherichia coli and mediates weak adhesion at low flow but strong adhesion at high flow. There is evidence that this occurs because FimH forms catch bonds, defined as bonds that are strengthened by tensile mechanical force. Here, we applied force to single isolated FimH bonds with an atomic force microscope in order to test this directly. If force was loaded slowly, most of the bonds broke up at low force (<60 piconewtons of rupture force). However, when force was loaded rapidly, all bonds survived until much higher force (140-180 piconewtons of rupture force), behavior that indicates a catch bond. Structural mutations or pretreatment with a monoclonal antibody, both of which allosterically stabilize a high affinity conformation of FimH, cause all bonds to survive until high forces regardless of the rate at which force is applied. Pretreatment of FimH bonds with intermediate force has the same strengthening effect on the bonds. This demonstrates that FimH forms catch bonds and that tensile force induces an allosteric switch to the high affinity, strong binding conformation of the adhesin. The catch bond behavior of FimH, the amount of force needed to regulate FimH, and the allosteric mechanism all provide insight into how bacteria bind and form biofilms in fluid flow. Additionally, these observations may provide a means for designing antiadhesive mechanisms.     

Aging-Related Differences in Chondrocyte Viscoelastic Properties

The biomechanical properties of articular cartilage change profoundly with aging. These changes have been linked with increased potential for cartilage degeneration and osteoarthritis. However, less is known about the change in biomechanical properties of chondrocytes with increasing age. Cell stiffness can affect mechanotransduction pathways and may alter cell function. We measured aging-related changes in the biomechanical properties of chondrocytes. Human chondrocytes were isolated from knee articular cartilage within 48 hours after death or from osteochondral specimens obtained from knee arthroplasty. Cells were divided into two age groups: between 18 and 35 years (18 -- 35); and greater than 55 years (55+) of age. The 55+ group was further subdivided based on visual grade of osteoarthritis: normal (N) or osteoarthritic (OA). The viscoelastic properties of the cell were measured using the previously described micropipette cell aspiration technique. The equilibrium modulus, instantaneous modulus, and apparent viscosity were significantly higher in the 55+ year age group than in the 18 -- 35 age group. On the other hand, no differences were found in the equilibrium modulus, instantaneous modulus, or apparent viscosity between the N and OA groups. The increase in cell stiffness can be attributed to altered mechanical properties of the cell membrane, the cytoplasm, or the cytoskeleton. Increased stiffness has been reported in osteoarthritic chondrocytes, which in turn has been attributed to the actin cytoskeleton. A similar mechanism may be responsible for our finding of increased stiffness in aging chondrocytes. With advancing age, changes in the biomechanical properties of the cell could alter molecular and biochemical responses.