Effects Of Mechanical Stretch Research Articles

Role of STIM1 in Stretch-induced Signaling in Human Airway Smooth Muscle.

Alteration in the normal mechanical forces of breathing can contribute to changes in contractility and remodeling characteristic of airway diseases, but the mechanisms that mediate these effects in airway cells are still under investigation. Airway smooth muscle (ASM) cells contribute to both contractility and extracellular matrix (ECM) remodeling. In this study, we explored ASM mechanisms activated by mechanical stretch, focusing on mechanosensitive piezo channels and the key Ca2+ regulatory protein stromal interaction molecule 1 (STIM1). Expression of Ca2+ regulatory proteins, including STIM1, Orai1 and caveolin-1, mechanosensitive ion channels Piezo-1 and Piezo-2, and NLRP3 inflammasomes were upregulated by 10% static stretch superimposed on 5% cyclic stretch. These effects were blunted by STIM1 siRNA. Histamine-induced [Ca2+]i responses and inflammasome activation were similarly blunted by STIM1 knockdown. These data show that the effects of mechanical stretch in human ASM cells are mediated through STIM1, which activates multiple pathways including Piezo channels and the inflammasome, leading to potential downstream changes in contractility and ECM remodeling.

High-magnitude cyclic stretch induces protein sialylation and RNA de-sialylation at the alveolar epithelial membrane

Sialylated glycans such as sialoglycoproteins, sialoglycolipids and sialoglycoRNA are ubiquitous in the glycocalyx of cells where they play a key role in barrier function, mechanosensation, infection, immune cell traffcking, and inflammation. These functions are particularly relevant at the blood-gas barrier of the lung. The alveolar epithelial glycocalyx is furthermore unique in that it is constantly exposed to cyclic stretch because of normal breathing. Yet, the abundance, regulation and function of sialylated glycans in the alveolar epithelial glycocalyx remains unknown. Here, we focused on the effects of mechanical stretch on the abundance and de novo formation of sialoglyco-proteins and -RNA as major constituents of the alveolar epithelial sialome. Human primary alveolar epithelial cells (hPAEpiC) were exposed to high magnitude cyclic stretch (18% CS) at 0.25 Hz for 24-48 hours to mimic biomechanical forces at the alveolar epithelium during high tidal volume ventilation. Total sialic acids in sialoglycoproteins were labeled with biotin by periodate oxidation and aldehyde ligation (pAL), and total sialic acids in sialoglycoRNA were quantified by 1, 2-diamino-4,5-methylene dioxybenzene ( DMB) assay. De novo formed sialic acids were labeled with biotin using N-azidoacetyl-mannosamine (Ac4ManNAz). Biotinylated siaolglyco-proteins and -RNAs were detected with streptavidin by Western and Northern blots, respectively, and quantified densitometrically. Stretch-dependent changes in the genes regulating sialic acid biosynthesis were assessed by RNA sequencing. High magnitude cyclic stretch increased the sialylation of proteins of different sizes (35-190 kDa) in the hPAEpiC glycocalyx. MGE showed this increase to be attributable to an increased de novo synthesis of sialic acids. On the other hand, high magnitude cyclic stretch reduced the sialylation of hPAEpiC sialoglycoRNA with no alteration in de novo synthesis, suggesting shedding, release or loss of sialoglycoRNA. RNA seq of hPAEpiC revealed that cyclic stretch upregulated the gene GNE (which encodes the enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase), the master regulator of sialic acid biosynthesis. Here, we demonstrate cyclic stretch-induced sialylation of proteins and de-sialylation of RNA in the alveolar epithelial glycocalyx. Given that sialic acids promote the adhesion of both infectious pathogens and immune cells, these effects may contribute to the pathogenesis of ventilator-associated pneumonia and ventilator-induced lung injury. This work is supported by the German Research Foundation, CRC 1449 subproject B01 to MO & WMK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of Mechanical Stretch on Sex-Dependent Gene Expression by RNA Sequencing in Mouse Renal Endothelial Cells

Increased mechanical endothelial cell stretch contributes to the development of numerous cardiovascular and renal pathologies by orchestration of immune cell activation. While endothelial cells are not recognized as professional antigen presenting cells, they can express both major histocompatibility complexes (MHC) I and II. MHCs are essential to form part of the immunological synapse during antigen presentation to adaptive immune cells. The mechanisms by which increased endothelial cell stretch regulates renal endothelial antigen processing and presentation is unknown. We hypothesized that increased mechanical endothelial stretch promotes upregulation of key antigen processing and presentation genes in mouse renal endothelial cells (MRECs) in a sex-dependent manner. MRECs derived from both sexes underwent 5%, 10%, and 15% (1 Hz) uniaxial cyclical stretch for 48 hours. After stretch, RNA sequencing was performed and bioinformatic and pathway analysis was executed to determine potential inflammatory mechanisms of action. We found that 10% stretch induced downregulation in MHC and costimulatory genes ( H2-Da, H2-K1, H2-T23, H2-T24) and upregulation of antigen processing associated genes ( tap1, psmb2, psmb4, and psmb8) in male MRECs. Females undergoing 10% stretch exhibited inverse gene expression profiles with upregulation of MHC genes and downregulation of antigen processing and presentation genes. At 15% stretch, the sex-specific gene expression tendencies were inverse from 10% stretch. To understand the function of differential gene expression we performed gene ontology enrichment pathway analysis. Male MRECs undergoing 10% stretch exhibited an enrichment for renal and cardiovascular development processes and inflammatory processes including chemokine-mediated signaling and cytokine-mediated signaling. When we compared female MRECs at 10% stretch, we found enrichment for renal and cardiovascular development processes including endothelial cell migration, angiogenesis and glomerular vascular, kidney, and artery development. Interestingly, 10% mechanical stretch enriched pro-inflammatory processes including positive regulation of cell migration and leukocyte migration. These data provide novel insight into how mechanical stretch regulates key antigen processing and presentation pathways in renal endothelial cells in a stretch and sex-dependent manner. This work was funded by MUSC Core Centers for Clinical Research Project Award to JPVB and Biomedical Laboratory Research and Development Service of the VA office of Research and Development Awards IK2BX003922 to KYDP and IK2BX005605 to JPVB. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mechanical Stretch Promotes Neurite Outgrowth of Primary Cultured Dorsal Root Ganglion Neurons via Suppression of Semaphorin 3A-Neuropilin-1/Plexin-A1 Signaling.

Significant attempts have been made to promote neuronal extension and migration in nerve development and regeneration. Although mechanical stretch induces persistent elongation of the axon, the underlying molecular mechanisms are not yet clear. Some axonal guidance cues secreted in the growth cone that affect the axonal growth could attract or repel axons in neurite connection. As semaphorin 3A (Sema3A) is an important repulsion guidance molecule, inhibition of Sema3A has been postulated to promote neuronal development. In this study, the effects of mechanical stretch on dorsal root ganglion neuronal growth and the underlying mechanisms were investigated by assessing the extension direction, neurite length, cell body size, mitochondrial membrane potential, and the expression of Sema3A and its receptors. Our results showed that cell viability significantly increased at tensile strains of 2.5, 5, and 10% for 4 h, with the most prominent effect at 5% tensile strain. Moreover, neurons migrated closer to the stretching direction at 5% tensile strain (0-12 h), while the neurons of the control group moved in a disorderly manner. Furthermore, Sema3A-Neuropilin-1/Plexin-A1 signaling pathway was found to be suppressed after mechanical stretch at 5% tensile strain for 4 h by immunofluorescence staining, immunoprecipitation, and western blot assay. Finally, a Sema3A-SiRNA (SiRNA = small interfering RNA) treatment led to remarkable guidance growth in the stretch-grown neurons. Importantly, there was significant decrease of repulsive cue Sema3A expression and remarkable increase of attractive molecule Netrin-1 expression after mechanical stretching treatment, which jointly promoted neurite outgrowth. This study provides a promising new approach for the development of mechanical stretching therapy or guidance factor-related drugs in injured neuronal regeneration.

The Effects of Mechanical Stretch on Integrins and Filopodial-Associated Proteins in Normal and Glaucomatous Trabecular Meshwork Cells.

The trabecular meshwork (TM) is the tissue responsible for regulating aqueous humor fluid egress from the anterior eye. If drainage is impaired, intraocular pressure (IOP) becomes elevated, which is a primary risk factor for primary open angle glaucoma. TM cells sense elevated IOP via changes in their biomechanical environment. Filopodia cellular protrusions and integrin transmembrane proteins may play roles in detecting IOP elevation, yet this has not been studied in detail in the TM. Here, we investigate integrins and filopodial proteins, such as myosin-X (Myo10), in response to mechanical stretch, an in vitro technique that produces mechanical alterations mimicking elevated IOP. Pull-down assays showed Myo10 binding to α5 but not the β1 subunit, αvβ3, and αvβ5 integrins. Several of these integrins colocalized in nascent adhesions in the filopodial tip and shaft. Using conformation-specific antibodies, we found that β1 integrin, but not α5 or αvβ3 integrins, were activated following 1-h mechanical stretch. Cadherin -11 (CDH11), a cell adhesion molecule, did not bind to Myo10, but was associated with filopodia. Interestingly, CDH11 was downregulated on the TM cell surface following 1-h mechanical stretch. In glaucoma cells, CDH11 protein levels were increased. Finally, mechanical stretch caused a small, yet significant increase in Myo10 protein levels in glaucoma cells, but did not affect cellular communication of fluorescent vesicles via filopodia-like tunneling nanotubes. Together, these data suggest that TM cell adhesion proteins, β1 integrin and CDH11, have relatively rapid responses to mechanical stretch, which suggests a central role in sensing changes in IOP elevation in situ.

PDGFR-β signaling mediates HMGB1 release in mechanically stressed vascular smooth muscle cells

Mechanically stressed vascular smooth muscle cells (VSMCs) have potential roles in the development of vascular complications. However, the underlying mechanisms are unclear. Using VSMCs cultured from rat thoracic aorta explants, we investigated the effects of mechanical stretch (MS) on the cellular secretion of high mobility group box 1 (HMGB1), a major damage-associated molecular pattern that mediates vascular complications in stressed vasculature. Enzyme-linked immunosorbent assay (ELISA) demonstrated an increase in the secretion of HMGB1 in VSMCs stimulated with MS (0-3% strain, 60 cycles/min), and this secretion was markedly and time-dependently increased at 3% MS. The increased secretion of HMGB1 at 3% MS was accompanied by an increased cytosolic translocation of nuclear HMGB1; the acetylated and phosphorylated forms of this protein were significantly increased. Among various inhibitors of membrane receptors mediating mechanical signals, AG1295 (a platelet-derived growth factor receptor (PDGFR) inhibitor) attenuated MS-induced HMGB1 secretion. Inhibitors of other receptors, including epidermal growth factor, insulin-like growth factor, and fibroblast growth factor receptors, did not inhibit this secretion. Additionally, MS-induced HMGB1 secretion was markedly attenuated in PDGFR-β-deficient cells but not in cells transfected with PDGFR-α siRNA. Likewise, PDGF-DD, but not PDGF-AA, directly increased HMGB1 secretion in VSMCs, indicating a pivotal role of PDGFR-β signaling in the secretion of this protein in VSMCs. Thus, targeting PDGFR-β-mediated secretion of HMGB1 in VSMCs might be a promising therapeutic strategy for vascular complications associated with hypertension.

The Effect of Mechanical Stretch on Myotube Growth Suppression by Colon-26 Tumor-Derived Factors.

Preclinical models and in vitro experiments have provided valuable insight into the regulation of cancer-induced muscle wasting. Colon-26 (C26) tumor cells induce cachexia in mice, and conditioned media (CM) from these cells promotes myotube atrophy and catabolic signaling. While mechanical stimuli can prevent some effects of tumor-derived factors on myotubes, the impact of mechanical signaling on tumor-derived factor regulation of myosin heavy chain (MyHC) expression is not well understood. Therefore, we examined the effects of stretch-induced mechanical signaling on C2C12 myotube growth and MyHC expression after C26 CM exposure. C26 CM was administered to myotubes on day 5 of differentiation for 48 h. During the last 4 or 24 h of C26 CM exposure, 5% static uniaxial stretch was administered. C26 CM suppressed myotube growth and MyHC protein and mRNA expression. Stretch for 24 h increased myotube size and prevented the C26 CM suppression of MyHC-Fast protein expression. Stretch did not change suppressed MyHC mRNA expression. Stretch for 24 h reduced Atrogin-1/MAFbx, MuRF-1, and LC3B II/I ratio and increased integrin β1D protein expression and the myogenin-to-MyoD protein ratio. Stretch in the last 4 h of CM increased ERK1/2 phosphorylation but did not alter the CM induction of STAT3 or p38 phosphorylation. These results provide evidence that in myotubes pre-incubated with CM, the induction of mechanical signaling can still provide a growth stimulus and preserve MyHC-Fast protein expression independent of changes in mRNA expression.

The Effect of Mechanical Stretch on Myotube Growth Suppression by Tumor‐derived Factors

The mechanisms of cancer-induced muscle wasting are widely investigated with in vivo preclinical models and in vitro experiments examining effects of tumor-derived factors. Myogenic differentiation is associated with rapid increases in myotube size and contractile properties, which tumor-derived factors can suppress. Specifically, colon-26 (C26) conditioned media induces catabolic signaling in cultured myotubes. While mechanical stimuli can induce anabolic signaling and myotube growth, the effect of mechanical stretch on myotubes treated with C26 conditioned media (CM) is not known. Therefore, we examined if uniaxial mechanical stretch could reverse myotube growth suppression caused by C26 conditioned media. Cachectic C26 or non-cachectic EL4 lymphoma CM was collected after 48h (~90% confluence). C2C12 myoblasts were seeded on type I collagen-coated polystyrene or Flexcell uniaxial stretch plates, proliferated, then placed in differentiation media for 5-7 days. On day 5 of differentiation, myotubes were treated with 50% control, C26, or EL4 CM for 48h. An additional experiment stretched myotubes 5% for the last 24h of the C26 incubation, and myotubes were collected immediately post stretch. Myotube diameter quantification used digital images. For all comparisons, at least three replicates from 2 independent experiments were used. Myotubes increased diameter (28%, p<0.0001) and myosin heavy chain-fast (MHC-F) protein expression (IOD/ug protein; 127%, p<0.0001) between days 5 and 7 of differentiation. Myotube growth was associated with increased AktT308 phosphorylation and suppressed E3 ligase expression. C26, but not EL4 suppressed myotube diameter (C26: -18%, p<0.0001; EL4: 1%, p=0.708) and MHC-F protein expression (C26: -62%, p<0.0001; EL4: 0%, p=0.999). C26 suppressed AktS473, ERK1/2, and 4E-BP1phosphorylation and increased MuRF1 and Atrogin1 expression. The C26 decrease in myotube diameter was not rescued by 24h of stretch (C26 control: -18%, p<0.0001; C26 stretch: -12%, p=0.0005). Stretch prevented C26 suppression of MHC-F protein expression (C26 control: -18%, p=0.001; C26 stretch: 11%, p=0.389). In summary, MHC-F protein concentration was rescued by mechanical stretch in C26 treated myotubes independent of myotube size. Additional research is warranted to determine how mechanical stretch prevents myosin's catabolic regulation in myotubes by tumor-derived factors.

Recent Advances in Maturation of Pluripotent Stem Cell-Derived Cardiomyocytes Promoted by Mechanical Stretch.

Stem cells have significant potential use in tissue regeneration, especially for treating cardiac diseases because of their multi-directional differentiation capability. By mimicking the in vivo physiological environment of native cardiomyocytes during their development and maturation, researchers have been able to induce pluripotent stem cell-derived cardiomyocytes (PSC-CMs) at high purity. However, the phenotype of these PSC-CMs is immature compared with that of adult cardiomyocytes. Various strategies have been explored to improve the maturity of PSC-CMs, such as long-term culturing, mechanical stimuli, chemical stimuli, and combinations of these strategies. Among these strategies, mechanical stretch as a key mechanical stimulus plays an important role in PSC-CM maturation. In this review, the optimal parameters of mechanical stretch, the effects of mechanical stretch on maturation of PSC-CMs, underlying molecular mechanisms as well as existing problems are discussed. Mechanical stretch is a powerful approach to promote the maturation of SC-CMs in terms of morphology, structure, and functionality. Nonetheless, further research efforts are needed to reach a satisfactory standard for clinical applications of PSC-CMs in treating cardiac diseases.

The Role of Hyperpolarization‐Activated Cyclic Nucleotide‐Gated Channels in Ileus Development

IntroductionIleus, a decrease or cessation of intestinal contractile activity, affects millions of trauma, post‐operative, and critically ill patients, resulting in longer hospital and ICU stays and increased patient complications. Mechanosensitive ion channels play a role in both the physiological and pathological responses to mechanical stimuli. We found that hyperpolarization‐activated cyclic nucleotide‐gated channel 2 (HCN2) was down‐regulated in edematous intestinal smooth muscle layers. However, the effects of altered HCN channel activity on intestinal contractile activity are largely unknown.ObjectiveThe purpose of the study was to investigate the effects of HCN inhibition on intestinal contractile activity. We hypothesize that inhibition of HCN will decrease intestinal contractile activity. Furthermore, we hypothesize that HCN activity is inhibited by mechanical stretch and/or inflammatory mediator changes that occur during the development of ileus.MethodsThe effects of HCN inhibitors on intestinal contractile activity were investigated ex vivo in an organ bath system. The effects of mechanical stretch, as would occur during the development of ileus, were subsequently examined. The effects of CXCL1, an inflammatory mediator shown to increase in both animal models of ileus and in trauma patients with ileus, were also examined.ResultsHCN inhibition with ZD7288 suppressed agonist‐induced intestinal contractile activity in a dose‐dependent, tetrodotoxin‐resistant manner. Inhibition with an alternative HCN inhibitor, Zatebradine, inhibited agonist‐induced contractile activity in a similar manner. HCN inhibition suppressed calcium sensitivity in the small intestinal. Interestingly, HCN inhibition did not inhibit spontaneous contractile activity. Subjecting intestinal tissue to increased stretch or pretreatment with CXCL1 reduced the effects of ZD7288 on intestinal contractile activity, suggesting that both mechanical stretch and inflammation reduce HCN activity. Preliminary data show that increased intestinal wall stretch decreased HCN protein levels.ConclusionsThe effects of HCN inhibition are tetrodotoxin‐resistant; thus, the effects of HCN inhibition are mediated in the smooth muscle, not the enteric nervous system. Both mechanical stretch and inflammation, which occur during the development of ileus, can inhibit HCN activity. Taken together, these data show that HCN channels are important in agonist‐induced intestinal contractile activity. HCN channels are a novel potential target in the treatment of ileus in post‐operative or critically ill patients.Support or Funding InformationHungarian National Research, Development, and Innovation Office (NKFI 120669) and Institutional Developments for Enhancing Intelligent Specialization Grant (EFOP‐3.6.2‐16‐2017‐0006)

Effects of stretching on molecular transfer from cell membrane by forming pores

ABSTRACTCell function was incessantly regulated by several microenvironments that were dependent on chemical and physical interactions. Physical forces, such as compressive stress, shear stress, and tensile forces, were transduced to biochemical signals through a multistep mechanotransduction process. Cell membrane proteins, such as cell membrane transporters and focal adhesions, cytoskeletal networks, and the cell membrane lipid bilayer, were among the primary targets for mechanical stimuli. Mechanical stretching was studied and applied for regenerative medicine and cellular therapies. To divulge the stretch loading’s working mechanism, a molecular dynamics simulation was conducted to apply the stretch loading to a 10 × 10 nm2 phosphatidylcholine (POPC) membrane bilayer. For this purpose, the LAMMPS computational package was used for implementing simulations. Under this state of mechanical loading, the electrical property alterations in the phospholipid bilayer (outer surface) and the membrane pore formation were studied. To validate the data that were obtained from the simulations, an experiment was designed to assess gene transfection in cells under the application of stretch loading. The pEGFP-N1 plasmid, encoding the Green Fluorescent Protein (GFP), is utilized as the gene transfection marker. The molecular dynamic results show that the stretch loading engendered a change in the membrane electrical charge; changes the outer surface charges of the phospholipid bilayer from negative to positive. In addition, the results indicate that mechanical stretching was involved in creating cell membrane pores. The experimental data confirmed the exclusive effect of mechanical stretch on the delivery of negatively charged DNA plasmid into the living cells. These results suggest that the electrical alteration at the membrane interface played an imperative role in the mechanotransduction downstream physiological responses.

The effects of mechanical stretch on the biological characteristics of human adipose-derived stem cells.

Adipose‐derived stem cells (ADSCs) are a subset of mesenchymal stem cells (MSCs), which have promised a vast therapeutic potential in tissue regeneration. Recent studies have demonstrated that combining stem cells with mechanical stretch may strengthen the efficacy of regenerative therapies. However, the exact influences of mechanical stretch on MSCs still remain inconclusive. In this study, human ADSCs (hADSCs) were applied cyclic stretch stimulation under an in vitro stretching model for designated duration. We found that mechanical stretch significantly promoted the proliferation, adhesion and migration of hADSCs, suppressing cellular apoptosis and increasing the production of pro‐healing cytokines. For differentiation of hADSCs, mechanical stretch inhibited adipogenesis, but enhanced osteogenesis. Long‐term stretch could promote ageing of hADSCs, but did not alter the cell size and typical immunophenotypic characteristics. Furthermore, we revealed that PI3K/AKT and MAPK pathways might participate in the effects of mechanical stretch on the biological characteristics of hADSCs. Taken together, mechanical stretch is an effective strategy for enhancing stem cell behaviour and regulating stem cell fate. The synergy between hADSCs and mechanical stretch would most likely facilitate tissue regeneration and promote the development of stem cell therapy.

Effect of Mechanical Stretch on the DNCB-induced Proinflammatory Cytokine Secretion in Human Keratinocytes

Skin is exposed to various physico-chemical cues. Keratinocytes, a major component of the skin epidermis, directly interact with the surrounding extracellular matrix, and thus, biochemical and biophysical stimulations from the matrix regulate the function of keratinocytes. Although it was reported that inflammatory responses of skin were altered by an applied mechanical force, understanding how the keratinocytes sense the mechanical stimuli and regulate a cytokine secretion remains unclear. Here, we designed a device that is able to apply chemo-mechanical cues to keratinocytes and assess their proinflammatory cytokine IL-6 production. We showed that when chemical stimuli were applied with mechanical stimuli simultaneously, the IL-6 production markedly increased compared to that observed with a single stimulus. Quantitative structural analysis of cellular components revealed that the applied mechanical stretch transformed the cell morphology into an elongated shape, increased the cell size, and dictated the distribution of focal adhesion complex. Our results suggest that the mechanical cue-mediated modulation of focal adhesion proteins and actin cytoskeleton translates into intracellular signaling associated with the IL-6 production particularly in skin sensitization. Our study can be applied to understand proinflammatory responses of skin under altered biophysical environments of the skin.

Intercondylar and central regions of complete discoid lateral meniscus have different cell and matrix organizations

BackgroundA complete discoid lateral meniscus (DLM) has a high risk of horizontal tear. However, cellular phenotypes and extracellular matrix organizations in complete DLMs are still unclear. The aim of this study was to investigate histological and cellular biological characteristics in both the intercondylar and central regions of complete DLM. Materials and methodsMeniscal samples were obtained from the intercondylar and central regions of complete DLM (n = 6). Blood vessels and aggregated cell ratio were measured in each region. Depositions of type I/II collagens and safranin O-stained proteoglycans in the extracellular matrix were assessed. Experiments in gene expression, morphology, proliferation, and effect of mechanical stretch were performed using cultured cells derived from each region. ResultsBlood vessel counts were significantly higher in the intercondylar region than in the central region. The ratio of aggregated cells was lower in the intercondylar region than in the central region. Deposition of type I collagen was comparable for both regions. The central region contained a larger quantity of type II collagen and safranin O staining density compared with the intercondylar region. Proliferation of the fibroblastic intercondylar cells was not affected by 5%-stretching. However, stretching treatments decreased relative proliferation of the chondrocytic central cells. ConclusionsThis study demonstrated that the central region of complete DLM had different cellular properties and collagen components compared with the intercondylar region. Our results suggest that the central region of complete DLM may have a low healing potential like the inner avascular region of the meniscus.

GW28-e0996 Mechanical Stretch induce Inflammatory Factors Expression and Pro-fibrotic Effect in Neonatal Rat Cardiac Fibroblast

To explore the effects of mechanical stretch on inflammatory cytokine expression and pro-fibrotic role in neonatal rat cardiac fibroblast. Neonatal rat cardiac fibroblasts(CFs) were obtained through differential adhesion method. The 2nd passage cells were stimulated by FLEXCELL-5000T system.

Real-time imaging of mechanically and chemically induced ATP release in human lung fibroblasts

Extracellular adenosine 5′-triphosphate (ATP) acts as an inflammatory mediator of pulmonary fibrosis. We investigated the effects of mechanical and chemical stimuli on ATP release from primary normal human lung fibroblasts. We visualized the ATP release from fibroblasts in real time using a luminescence imaging system while acquiring differential interference contrast cell images with infrared optics. Immediately following a single uniaxial stretch for 1s, ATP was released from a certain population of cells and spread to surrounding spaces. Hypotonic stress, which causes plasma membrane stretching, also induced the ATP release. Compared with the effects of mechanical stretch, ATP-induced release sites were homogeneously distributed. In contrast to the effects of mechanical stimuli, application of platelet-derived growth factor caused ATP release from small numbers of the cells. Our real-time ATP imaging demonstrates that there is a heterogeneous nature of ATP release from lung fibroblasts in response to mechanical and chemical stimuli.

Mechanical Loading Improves Tendon-Bone Healing in a Rabbit Anterior Cruciate Ligament Reconstruction Model by Promoting Proliferation and Matrix Formation of Mesenchymal Stem Cells and Tendon Cells

Background/Aims: This study investigated the effect of mechanical stress on tendon-bone healing in a rabbit anterior cruciate ligament (ACL) reconstruction model as well as cell proliferation and matrix formation in co-culture of bone-marrow mesenchymal stem cells (BMSCs) and tendon cells (TCs). Methods: The effect of continuous passive motion (CPM) therapy on tendon-bone healing in a rabbit ACL reconstruction model was evaluated by histological analysis, biomechanical testing and gene expressions at the tendon-bone interface. Furthermore, the effect of mechanical stretch on cell proliferation and matrix synthesis in BMSC/TC co-culture was also examined. Results: Postoperative CPM therapy significantly enhanced tendon-bone healing, as evidenced by increased amount of fibrocartilage, elevated ultimate load to failure levels, and up-regulated gene expressions of Collagen I, alkaline phosphatase, osteopontin, Tenascin C and tenomodulin at the tendon-bone junction. In addition, BMSC/TC co-culture treated with mechanical stretch showed a higher rate of cell proliferation and enhanced expressions of Collagen I, Collagen III, alkaline phosphatase, osteopontin, Tenascin C and tenomodulin than that of controls. Conclusion: These results demonstrated that proliferation and differentiation of local precursor cells could be enhanced by mechanical stimulation, which results in enhanced regenerative potential of BMSCs and TCs in tendon-bone healing.

Mechanical stretch and hypoxia inducible factor-1 alpha affect the vascular endothelial growth factor and the connective tissue growth factor in cultured ACL fibroblasts

ABSTRACTPurposes: The adult human anterior cruciate ligament (ACL) has poor functional healing response. Hypoxia plays an important role in regulating the microenvironment of the joint cavity after ACL injury, however, its role in mechanical injury is yet to be examined fully in ACL fibroblasts. In this study, we used CoCl2 to induce Hypoxia-inducible factor-1α (HIF-1α) in our experimental model to study its affect on matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) expression in ACL fibroblasts after mechanical stretch. Materials and methods: Cell treatments were performed in the stretch chamber in all experimental groups. Quantitative real-time PCR was used to check mRNA expression levels of MMP-2, CTGF, VEGF, and HIF-1α. Western blot was used to detect the HIF-1α production. Enzyme-Linked immunosorbent assay was performed to check the VEGF and CTGF protein contents in supernatant. MMP-2 activity was assayed by gelatin zymography. Results: The real-time PCR results show that mechanical stretch or CoCl2 treatment increases the expression of MMP-2, VEGF, CTGF, and HIF-1α; however, the combined effects of mechanical stretch and CoCl2-induced HIF-1α increased MMP-2 production but decreased the VEGF and CTGF expression, compared to the CoCl2 treatment group alone. Western blot analysis and ELISA also confirmed these results. Conclusions: Our results demonstrated that mechanical stretch and CoCl2-induced HIF-1α together increased the level of MMP-2 and decreased the levels of VEGF and CTGF in cultured ACL fibroblasts. The differential expression and production of HIF-1α, VEGF, MMP-2, and CTGF might help to explain the poor healing ability of ACL.

Effects of Mechanical Stretch on Cell Proliferation and Matrix Formation of Mesenchymal Stem Cell and Anterior Cruciate Ligament Fibroblast.

Mesenchymal stem cells (MSCs) and fibroblasts are two major seed cells for ligament tissue engineering. To understand the effects of mechanical stimulation on these cells and to develop effective approaches for cell therapy, it is necessary to investigate the biological effects of various mechanical loading conditions on cells. In this study, fibroblasts and MSCs were tested and compared under a novel Uniflex/Bioflex culture system that might mimic mechanical strain in ligament tissue. The cells were uniaxially or radially stretched with different strains (5%, 10%, and 15%) at 0.1, 0.5, and 1.0 Hz. The cell proliferation and collagen production were compared to find the optimal parameters. The results indicated that uniaxial stretch (15% at 0.5 Hz; 10% at 1.0 Hz) showed positive effects on fibroblast. The uniaxial strains (5%, 10%, and 15%) at 0.5 Hz and 10% strain at 1.0 Hz were favorable for MSCs. Radial strain did not have significant effect on fibroblast. On the contrary, the radial strains (5%, 10%, and 15%) at 0.1 Hz had positive effects on MSCs. This study suggested that fibroblasts and MSCs had their own appropriate mechanical stimulatory parameters. These specific parameters potentially provide fundamental knowledge for future cell-based ligament regeneration.

Enhancement of Automaticity by Mechanical Stretch of the Isolated Guinea Pig Pulmonary Vein Myocardium.

The effects of mechanical stretch on the automaticity of the guinea pig isolated pulmonary vein preparations were studied in microelectrode experiments. The application of cumulative mechanical stretch to the preparations resulted in increases in the firing rate of spontaneous electrical activity in the myocardial layer. This effect was significantly inhibited by stretch-activated channel blockers such as gadolinium or streptomycin. These results suggest that acute mechanical stretch enhances the automaticity of the guinea pig pulmonary vein myocardium through the opening of the stretch-activated channels.