Cyclic stretch enhances neutrophil extracellular trap formation.

BACKGROUND: Neutrophils, the most abundant leukocytes circulating in blood, contribute to host defense and play a significant role in chronic inflammatory disorders. They can release their DNA in the form of extracellular traps (NETs), which serve as scaffolds for capturing bacteria and various blood cells. However, uncontrolled formation of NETs (NETosis) can lead to excessive activation of coagulation pathways and thrombosis. Once neutrophils are migrated to infected or injured tissues, they become exposed to mechanical forces from their surrounding environment. However, the impact of transient changes in tissue mechanics due to the natural process of aging, infection, tissue injury, and cancer on neutrophils remains unknown. To address this gap, we explored the interactive effects of changes in substrate stiffness and cyclic stretch on NETosis. Primary neutrophils were cultured on a silicon-based substrate with stiffness levels of 30 and 300 kPa for at least 3 h under static conditions or cyclic stretch levels of 5% and 10%, mirroring the biomechanics of aged and young arteries. RESULTS: Using this approach, we found that neutrophils are sensitive to cyclic stretch and that increases in stretch intensity and substrate stiffness enhance nuclei decondensation and histone H3 citrullination (CitH3). In addition, stretch intensity and substrate stiffness promote the response of neutrophils to the NET-inducing agents phorbol 12-myristate 13-acetate (PMA), adenosine triphosphate (ATP), and lipopolysaccharides (LPS). Stretch-induced activation of neutrophils was dependent on calpain activity, the phosphatidylinositol 3-kinase (PI3K)/focal adhesion kinase (FAK) signalling and actin polymerization. CONCLUSIONS: In summary, these results demonstrate that the mechanical forces originating from the surrounding tissue influence NETosis, an important neutrophil function, and thus identify a potential novel therapeutic target.

Dual role of endoplasmic reticulum stress-ATF-6 activation in autophagy and apoptosis induced by cyclic stretch in myoblast.

OBJECTIVE: Mandibular growth that is induced by functional appliances is closely associated with skeletal and neuromuscular adaptation. Accumulating evidence has proved that apoptosis and autophagy have a vital role in adaptation process. However, little is known about the underlying mechanisms. This study sought to determine whether ATF-6 is involved in stretch-induced apoptosis and autophagy in myoblast. The study also sought to uncover the potential molecular mechanism. MATERIALS AND METHODS: Apoptosis was assessed by TUNEL and Annexin V and PI staining. Autophagy was detected by transmission electron microscopy (TEM) analysis and immunofluorescent staining for autophagy-related protein light chain 3 (LC3). Real time-PCR and western blot were performed to evaluate the expression level of mRNA and proteins that were associated with endoplasmic reticulum stress (ERS), autophagy and apoptosis. RESULTS: Cyclic stretch significantly decreased the cell viability and induced apoptosis and autophagy of myoblasts time-dependently. Stretching stimuli activated ATF-6 pathway and induced ERS-mediated apoptosis. Moreover, using 4-PBA significantly inhibited ERS-related apoptosis, as well as partially decreasing autophagy. In addition, inhibition of autophagy by 3-MA enhanced apoptosis by affecting the expression of CHOP and Bcl-2. However, it had no obvious effects on ERS-related proteins of GRP78 and ATF-6. More importantly, knockdown ATF-6 effectively weakened apoptosis and autophagy. It did so by regulating the expression of Bcl-2, Beclin1 and CHOP, but not cleaved Caspase-12, LC3II and p62 in stretched myoblast. CONCLUSION: ATF-6 pathway was activated in myoblast by mechanical stretch. ATF-6 may regulate the process of stretch-induced myoblast apoptosis and autophagy via CHOP, Bcl-2 and Beclin1 signaling.

The cellular responses of corneal fibroblasts to cyclic stretching loads.

Mechanical signaling plays a crucial role in maintaining extracellular matrix (ECM) homeostasis in various structures. In this study, we investigated the responses of corneal fibroblasts to cyclic stretching loads using an in vitro cell culture system. Bovine corneal fibroblasts were cultured and subjected to equibiaxial cyclic strain of 15% for 72 h at a frequency of 0.25 Hz, with bovine skin fibroblasts used as a comparison. We explored various cellular behaviors, including morphological changes, cell proliferation, and metabolism in response to mechanical stretching loads. The expression of genes, protein secretion, and enzymatic activity for several major metalloproteinases was also determined through Q-PCR, Western blot, and gel zymography. Additionally, we investigated the involvement of mitogen-activated protein kinases (MAPKs) signaling pathways in the corneal fibroblasts when subjected to mechanical stimuli. Our findings revealed that, compared to skin fibroblasts, corneal fibroblasts were reluctant to morphological changes in response to a prolonged (72 h) and high-amplitude (15% of strain) cyclic stretching load. However, cyclic stretching loads stimulated the upregulation of MMP-2 expression in corneal fibroblasts via the MAPK signaling pathways involving extracellular signal-regulated kinase and p38. Together with a lack of upregulation in type I collagen expression, our results indicate the induction of the ECM degradation process in corneal fibroblasts in response to cyclic stretching. These findings emphasize the mechanoresponsive nature of corneal fibroblasts and shed light on the potential impact of intense mechanical stress on the cornea in both normal and pathological conditions such as keratoconus, providing valuable insights for understanding corneal mechanobiology.

Inhibition of importin-7 attenuates ventilator-induced lung injury by targeting nuclear translocation of p38.

BACKGROUND: The ability of p38 to phosphorylate substrates in the nucleus and the role of nuclear p38 in the regulation of inflammation have focused attention on the subcellular localization of the kinase. Although it is clear that p38 shuttles to the nucleus upon stimulation, the mechanisms that regulate p38 nuclear input in response to mechanical stretch remain to be determined. METHODS: Cyclic stretch (CS)-induced nuclear translocation of p38 was determined by Western blotting and immunofluorescence. The p38 interacting protein was identified using endogenous pull-down and protein binding assays. The potential role of importin-7 (Imp7) in CS-induced nuclear translocation of p38 and p38-dependent gene expression was confirmed using a series of in vitro and in vivo experiments. Furthermore, we tested the therapeutic potential of intratracheal administration of Imp7 siRNA-loaded nanoparticles in the ventilator-induced lung injury (VILI) mouse model. RESULTS: We show that CS induced phosphorylation-dependent nuclear translocation of p38, which required the involvement of microtubules and dynein. Endogenous pull-down assay revealed Imp7 to be a potential p38-interacting protein, and the direct interaction between p38 and Imp7 was confirmed by in vitro and in vivo binding assays. Furthermore, silencing Imp7 inhibited CS-induced nuclear translocation of p38 and subsequent cytokine production. Notably, intratracheal administration of Imp7 siRNA nanoparticles attenuated lung inflammation and histological damage in the VILI mouse model. CONCLUSIONS: Our findings uncover a key role for Imp7 in the process of p38 nuclear import after CS stimulation and highlight the potential of preventing p38 nuclear translocation in treatment of VILI.

YAP/WNT5A/FZD4 axis regulates osteogenic differentiation of human periodontal ligament cells under cyclic stretch.

OBJECTIVE: To verify the role of YAP/WNT5A/FZD4 axis in stretch-induced osteogenic differentiation of hPDLCs. BACKGROUND: During orthodontic tooth movement, differentiation of human periodontal ligament cells (hPDLCs) at the tension side of the periodontal ligament mediates new bone formation. WNT5A promotes osteogenesis and its regulator Yes-associated protein (YAP) is responsive to mechanical stimulation in hPDLCs. However, the mechanisms of YAP and WNT5A in alveolar bone remodeling remain unclear. METHODS: Cyclic stretch was applied to hPDLCs to mimic the orthodontic stretching force. Osteogenic differentiation was determined by alkaline phosphatase (ALP) activity, Alizarin Red staining, qRT-PCR and western blotting. To detect activation of YAP and expression of WNT5A and its receptor Frizzled-4 (FZD4), western blotting, immunofluorescence, qRT-PCR and ELISA were performed. Verteporfin, Lats-IN-1, small interfering RNAs and recombinant protein were used to explore the relationship of YAP, WNT5A and FZD4, and the effect of their relationship on stretch-induced osteogenesis of hPDLCs. RESULTS: WNT5A, FZD4 and nuclear localization of YAP were upregulated by cyclic stretch. YAP positively regulated WNT5A and FZD4 expression and osteogenic differentiation of hPDLCs under cyclic stretch by YAP inhibition or activation assay. Knockdown of WNT5A and FZD4 attenuated YAP-induced and stretch-induced osteogenic differentiation. Recombinant WNT5A rescued the suppressed osteogenic differentiation by YAP inhibitor in hPDLCs, whereas knockdown of FZD4 weakened the effect of WNT5A and amplified the suppression. CONCLUSIONS: WNT5A/FZD4 could be positively regulated by YAP and the YAP/WNT5A/FZD4 axis mediated osteogenic differentiation of hPDLCs under cyclic stretch. This study provided further insight into the biological mechanism of orthodontic tooth movement.

Clinical Translation of Engineered Pulmonary Vascular Models.

Diseases in pulmonary vasculature remain a major cause of morbidity and mortality worldwide. Numerous pre-clinical animal models were developed to understand lung vasculature during diseases and development. However, these systems are typically limited in their ability to represent human pathophysiology for the study of disease and drug mechanisms. In recent years, a growing number of studies have focused on developing in vitro experimental platforms that mimic human tissues/organs. In this chapter, we discuss the key components involved in developing engineered pulmonary vascular modeling systems and provide perspectives on ways to improve the translational potential of existing models.

Silencing of Nesprin-2 inhibits the differentiation of myofibroblasts from fibroblasts induced by mechanical stretch.

Mechanical force plays a pivotal role in the pathogenesis of hypertrophic scar (HTS). Dermal fibroblasts and myofibroblasts are the key cells involved in HTS. Myofibroblasts in HTS possess different biochemical and biophysical characteristics by which myofibroblasts are often distinguished from fibroblasts. The role of mechanotransducers outside the nucleus in the pathogenesis of HTS has been reported in many studies. However, the role of Nesprin-2 in HTS is not clear. Hence, we aim to construct a cell model of HTS and explore the role of Nesprin-2 in this process. Myofibroblasts and fibroblasts were isolated from HTS and healthy skin tissues of the same patient. Fibroblasts were exposed to cyclic stretch with 10% magnitude and a frequency of 0.1 Hz for 3 days, 5 days, and 7 days, respectively. After the cell model was confirmed, fibroblasts transfected with siRNA targeting human Nesprin-2 were exposed to cyclic stretch. The mechanical behaviour and biochemical reaction of the dermal fibroblasts were analysed. The stretched fibroblasts at day 5 showed the same mechanotransductive and biochemical features as unstretched myofibroblasts. Mechanical strain could induce the myofibroblasts differentiation and a cell model of HTS was established successfully at day 5. The expressions of lamin A/C, alpha-smooth muscle actin, transforming growth factor beta 1, and collagen type I in fibroblasts were reduced by the silencing of Nesprin-2. Mechanical strain could induce the myofibroblasts differentiation and silencing of Nesprin-2 could block the mechanical stimulation of terminal myofibroblasts differentiation. Nesprin-2 might be a potential target to treat the HTS.

Mechanosensitive myosin II but not cofilin primarily contributes to cyclic cell stretch-induced selective disassembly of actin stress fibers.

Cells adapt to applied cyclic stretch (CS) to circumvent chronic activation of proinflammatory signaling. Currently, the molecular mechanism of the selective disassembly of actin stress fibers (SFs) in the stretch direction, which occurs at the early stage of the cellular response to CS, remains controversial. Here, we suggest that the mechanosensitive behavior of myosin II, a major cross-linker of SFs, primarily contributes to the directional disassembly of the actomyosin complex SFs in bovine vascular smooth muscle cells and human U2OS osteosarcoma cells. First, we identified that CS with a shortening phase that exceeds in speed the inherent contractile rate of individual SFs leads to the disassembly. To understand the biological basis, we investigated the effect of expressing myosin regulatory light-chain mutants and found that SFs with less actomyosin activities disassemble more promptly upon CS. We consequently created a minimal mathematical model that recapitulates the salient features of the direction-selective and threshold-triggered disassembly of SFs to show that disassembly or, more specifically, unbundling of the actomyosin bundle SFs is enhanced with sufficiently fast cell shortening. We further demonstrated that similar disassembly of SFs is inducible in the presence of an active LIM-kinase-1 mutant that deactivates cofilin, suggesting that cofilin is dispensable as opposed to a previously proposed mechanism.

Endothelial microparticles induced by cyclic stretch activate Src and modulate cell apoptosis.

Endothelial microparticles (EMPs) are involved in various cardiovascular pathologies and play remarkable roles in communication between endothelial cells (ECs), which are constantly exposed to mechanical cyclic stretch (CS) following blood pressure. However, the roles of EMPs induced by CS in EC homeostasis are still unclear. Both fluorescence resonance energy transfer (FRET) and western blotting revealed the activation of Src in ECs was significantly increased by 5% CS-induced EMPs. Furthermore, proteomic analysis revealed that the contents were obvious different in the EMPs between 5%- and 15%-group. Based on the bioinformatic analysis, CD151 on EMPs was predicted to activate Src, which was further confirmed by both FRET and western blotting. Moreover, the expression of CD151 on EMPs was significantly increased by 5% CS and involved in the binding of EMPs to ECs. EC apoptosis, which was significantly decreased by 5% CS-derived EMPs, showed obvious increase after pretreatment with Src inhibitor in target ECs. Our present research suggests that mechanical stretch changes the components of EMPs, which in turn modulates EC apoptosis by Src activation. CD151 expressed on CS-induced EMPs may play important roles in EC communication and homeostasis.

Endothelial microvesicles induced by physiological cyclic stretch inhibit ICAM1-Dependent leukocyte adhesion.

Physiological cyclic stretch (CS), caused by artery deformation following blood pressure, plays important roles in the homeostasis of endothelial cells (ECs). Here, we detected the effect of physiological CS on endothelial microvesicles (EMVs) and their roles in leukocyte recruitment to ECs, which is a crucial event in EC inflammation. The results showed compared with the static treatment, pretreatment of 5%-CS-derived EMVs with ECs significantly decreased the adherence level of leukocytes. Comparative proteomic analysis revealed 373 proteins differentially expressed between static-derived and 5%-CS-derived EMVs, in which 314 proteins were uniquely identified in static-derived EMVs, 34 proteins uniquely in 5%-CS-derived EMVs, and 25 proteins showed obvious differences. Based on the proteomic data, Ingenuity Pathways Analysis predicted intercellular adhesion molecule 1 (ICAM1) in EMVs might be the potential molecule involved in EC-leukocyte adhesion. Western blot and flow cytometry analyses confirmed the significant decrease of ICAM1 in 5%-CS-derived EMVs, which subsequently inhibited the phosphorylation of VE-cadherin at Tyr731 in target ECs. Moreover, leukocyte adhesion was obviously decreased after pretreatment with ICAM1 neutralizing antibody. Our present research suggested that physiological stretch changes the components of EMVs, which in turn inhibits leukocyte adhesion. ICAM1 expressed on CS-induced EMVs may play an important role in maintaining EC homeostasis.

Surface Charge-Dependent Cytotoxicity of Plastic Nanoparticles in Alveolar Cells under Cyclic Stretches.

Polystyrene nanoparticles (PS-NPs) derived from both environmental and occupational sources are an important class of ultrafine particles associated with human pulmonary disorders. The effects of surface charges of particle internalization and toxicity to alveolar cells, especially under conditions comparable to those found during breathing, have not been examined. Here, we applied cyclic stretches (CS) to human alveolar cells during nanoparticle exposure and show an enhanced accumulation of positively charged polystyrene nanoparticles as compared to similar negatively charged particles. The cellular uptake of the positive particles into live cells was visualized with three-dimensional optical diffraction tomography (3-D ODT). The simultaneous application of both periodic stretching as well as positively charged nanoparticles led to blebbing morphology and activation of apoptotic signaling compared to control cells. Our findings provide a better understanding of how surface charge mediates the uptake and toxicity of nanoplastics under the dynamical mechanical conditions relevant for breathing exposures.

CTGF regulates cyclic stretch-induced vascular smooth muscle cell proliferation via microRNA-19b-3p.

Cyclic stretch regulates proliferation of vascular smooth muscle cells (VSMCs) during hypertension-induced vascular remodeling, but the underlying mechanisms remain to be studied. Connective tissue growth factor (CTGF) has been reported associated with several cellular function such as proliferation，migration and adhesion. Herein, the role of CTGF in VSMCs was investigated in response to mechanical cyclic stretch. Here we show that CTGF is up-regulated both in vivo and in vitro during hypertension. Overexpression of CTGF markedly promoted VSMC proliferation, whereas CTGF knockdown attenuated cyclic stretch-induced proliferation. Furthermore, 3'UTR reporter assays revealed that microRNA-19b-3p (miR-19b-3p) directly regulates CTGF expression. Under pathological condition (e.g. 15% cyclic stretch), miR-19b-3p expression was significantly down-regulated; conversely miR-19b-3p overexpression blocked VSMC proliferation. Taken together, these findings indicate that pathological cyclic stretch induces vascular remodeling by promoting VSMC proliferation via miR-19b-3p/CTGF pathway, and point to CTGF as a potential therapeutic target for hypertension.

MicroRNA-29a Involvement in Phenotypic Transformation of Venous Smooth Muscle Cells Via Ten-Eleven Translocation Methylcytosinedioxygenase 1 in Response to Mechanical Cyclic Stretch.

Mechanical stimuli play an important role in vein graft restenosis and the abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) are pathological processes contributing to this disorder. Here, based on previous high-throughput sequencing data from vein grafts, miR-29a-3p and its target, the role of Ten-eleven translocation methylcytosinedioxygenase 1 (TET1) in phenotypic transformation of VSMCs induced by mechanical stretch was investigated. Vein grafts were generated by using the "cuff" technique in rats. Deep transcriptome sequencing revealed that the expression of TET1 was significantly decreased, a process confirmed by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis. MicroRNA-seq showed that miR-29a-3p was significantly up-regulated, targeting TET1 as predicted by Targetscan. Bioinformatics analysis indicated that the co-expressed genes with TET1 might modulate VSMC contraction. Venous VSMCs exposed to 10%-1.25 Hz cyclic stretch by using the Flexcell system were used to simulate arterial mechanical conditions in vitro. RT-qPCR revealed that mechanical stretch increased the expression of miR-29a-3p at 3 h. Western blot analysis showed that TET1 was significantly decreased, switching contractile VSMCs to cells with a synthetic phenotype. miR-29a-3p mimics (MI) and inhibitor (IN) transfection confirmed the negative impact of miR-29a-3p on TET1. Taken together, results from this investigation demonstrate that mechanical stretch modulates venous VSMC phenotypic transformation via the mediation of the miR-29a-3p/TET1 signaling pathway. miR-29a-3p may have potential clinical implications in the pathogenesis of remodeling of vein graft restenosis.

High stretch cycling inhibits the morphological and biological decidual process in human endometrial stromal cells.

PURPOSE: Subendometrial myometrium exerts wave-like activity throughout the menstrual cycle, and uterine peristalsis is markedly reduced during the implantation phase. We hypothesized that abnormal uterine peristalsis has an adverse effect on the endometrial decidualization process. We conducted an in vitro culture experiment to investigate the effect of cyclic stretch on the morphological and biological endometrial decidual process. METHODS: Primary human endometrial stromal cells (HESCs) were isolated from hysterectomy specimens and incubated with or without 8-bromo-cyclic adenosine monophosphate (8-br-cAMP) and medroxyprogesterone acetate (MPA) for 3 days. After decidualization, cultures were continued for 24 hours with or without cyclic stretch using a computer-operated cell tension system. RESULTS: Cyclic stretch significantly repressed expression of decidual markers including insulin-like growth factor-binding protein 1 (IGFBP1), prolactin (PRL), forkhead box O1 (FOXO1), and WNT4 on decidualized HESCs. In addition, cyclic stretch of decidualized HESCs affected the decidual morphological phenotype to an elongated shape. The alternation of F-actin localization in decidualized HESCs was not observed in response to cyclic stretch. CONCLUSIONS: These data suggest that cyclic stretch inhibits the morphological and biological decidual process of HESCs. Our findings imply that uterine abnormal contractions during the implantation period impair endometrial decidualization and contribute to infertility.

Effect of atorvastatin on cardiomyocyte hypertrophy through suppressing MURC induced by volume overload and cyclic stretch.

MURC (muscle-restricted coiled-coil protein) is a hypertrophy-related gene. Hypertrophy can be induced by mechanical stress. The purpose of this research was to investigate the hypothesis that MURC mediates hypertrophy in cardiomyocytes under mechanical stress. We used the in vivo model of an aortocaval shunt (AV shunt) in adult Wistar rats to induce myocardial hypertrophy. We also used the in vitro model of cyclic stretch in rat neonatal cardiomyocytes to clarify MURC expression and the molecular regulation mechanism. The flexible membrane culture plate seeding with cardiomyocytes Cardiomyocytes seeded on a flexible membrane culture plate were stretched by vacuum pressure to 20% of maximum elongation at 60 cycles/min. AV shunt induction enhanced MURC protein expression in the left ventricular myocardium. Treatment with atorvastatin inhibited the hypertrophy induced by the AV shunt. Cyclic stretch markedly enhanced MURC protein and mRNA expression in cardiomyocytes. Addition of extracellular-signal-regulated kinase (ERK) inhibitor PD98059, ERK small interfering RNA (siRNA), angiotensin II (Ang II) antibody and atorvastatin before stretch, abolished the induction of MURC protein. An electrophoretic mobility shift assay showed that stretch enhanced the DNA binding activity of serum response factor. Stretch increased but MURC mutant plasmid, ERK siRNA, Ang II antibody and atorvastatin reversed the transcriptional activity of MURC induced by stretch. Adding Ang II to the cardiomyocytes also induced MURC protein expression. MURC siRNA and atorvastatin inhibited the hypertrophic marker and protein synthesis induced by stretch. Treatment with atorvastatin reversed MURC expression and hypertrophy under volume overload and cyclic stretch.

Interaction between caspase-3 and caspase-5 in the stretch-induced programmed cell death in the human periodontal ligament cells.

In our previous studies, programmed cell death (PCD) was induced in human periodontal ligament (PDL) cells, through activation of caspase-3 and upregulation of CASP5 gene (encoding caspase-5 protein), in response to mechanical stretch loading. The aim of this study is to explore the relationship between the inflammatory caspase, caspase-5, and the apoptotic executioner protein, caspase-3, in human PDL cells. Here, we found that cyclic stretching upregulated the activity and the protein expression level of caspase-3 and -5 and the addition of the caspase-3 inhibitor or caspase-5 inhibitor significantly inhibited the stretch-induced PCD. Meanwhile, the inhibition of caspase-5 inhibited the activation of caspase-3 and vice versa. The result of coimmunoprecipitation also demonstrated that the expression of caspase-3 was immunoprecipitated with caspase-5. Thus, our study revealed that the in vitro application of cyclic stretching induced PCD by activation of caspase-3 and -5 in human PDL cells, and these two caspases could interact with each other after mechanical stretch loading. The study may facilitate further studies on the mechanism of stretch-induced PCD and help us understand the force-related periodontal homeostasis and remodeling better.

MicroRNA-132-3p represses Smad5 in MC3T3-E1 osteoblastic cells under cyclic tensile stress.

MicroRNAs (miRNAs) regulate osteogenic differentiation of bone cells, which has applications in orthodontics. Here we evaluated the miRNA expression profile of MC3T3-E1 osteoblasts under cyclic tensile stress with chip technology and found that miR-132-3p was up-regulated by 12% cyclic tensile stress. Alkaline phosphatase activity and osteocalcin expression in MC3T3-E1 cells were decreased under these conditions. Smad2 and Smad5 were identified as potential target genes of miR-132-3p. Native and phosphorylated Smad2 and Smad5 expression was negatively correlated with miR-132-3p levels in the cells under cyclic stretch; however, only Smad5 protein level was reduced upon miR-132-3p overexpression. The luciferase reporter assay confirmed a direct interaction between miR-132-3p and Smad5. Thus, miR-132-3p maybe regulates osteoblast differentiation via Smad5 in response to cyclic tensile stress.

Cyclic stretch induced oxidative stress by mitochondrial and NADPH oxidase in retinal pigment epithelial cells.

BACKGROUND: Vitreomacular adhesion (VMA) has been reported to associated with age-related macular degeneration (AMD). Understanding the mechanisms underlying cyclic stretch induced in retinal pigment epithelial cells (RPE) may be important for the treatment of VMA-related AMD. METHOD: Cyclic stretch (1HZ, 20% elongation) was applied to cultured ARPE-19 cells for 15 min, 2 h, 6 h, 12 h, 24 h by flexcell FX-5000 Tension system. Total reactive oxygen species (ROS) were detected using DCFH-DA. Mitochondrial superoxide were detected using MitoSOX Red mitochondrial superoxide indicator. NADPH oxidases (NOX) and signaling pathways, such as p38 and PKC, were detected using western blot. Apocycin (Apo) were used as NOX inhibitors. RESULT: High levels of total ROS were detected from 15 min to 24 h, whereas mitochondrial superoxide were higher only in early time. NOX2 were significantly increased at 24 h. NOX4 were significantly increased at 2 h and reach its peak at 24 h. P-p38 was significantly increased at 12 h and 24 h. P-PKC was significantly increased at 15 min and kept a persistent high level. The upregulated expression of NOX4 by cyclic stretch can be significantly decreased under p-PKC inhibitor other than p-p38 inhibitor. CONCLUSION: Cyclic stretch induce oxidative stress from both mitochodrial and NADPH oxidase in RPE cells, which may prompt oxidative damage in VMA-related AMD.

Effect of Cyclic Stretch on Vascular Endothelial Cells and Abdominal Aortic Aneurysm (AAA): Role in the Inflammatory Response.

Abdominal aortic aneurysm (AAA) is a focal dilatation of the aorta, caused by both genetic and environmental factors. Although vascular endothelium plays a key role in AAA progression, the biological mechanisms underlying the mechanical stress involvement are only partially understood. In this study, we developed an in vitro model to characterize the role of mechanical stress as a potential trigger of endothelial deregulation in terms of inflammatory response bridging between endothelial cells (ECs), inflammatory cells, and matrix remodeling. In AAA patients, data revealed different degrees of calcification, inversely correlated with wall stretching and also with inflammation and extracellular matrix degradation. In order to study the role of mechanical stimulation, endothelial cell line (EA.hy926) has been cultured in healthy (10% strain) and pathological (5% strain) dynamic conditions using a bioreactor. In presence of tumor necrosis factor alpha (TNF-α), high levels of matrix metalloproteinase-9 (MMP-9) expression and inflammation are obtained, while mechanical stimulation significantly counteracts the TNF-α effects. Moreover, physiological deformation also plays a significant role in the control of the oxidative stress. Overall our findings indicate that, due to wall calcification, in AAA there is a significant change in terms of decreased wall stretching.

Sensory Neuropeptides and their Receptors Participate in Mechano-Regulation of Murine Macrophages.

This study aimed to analyze if the sensory neuropeptide SP (SP) and the neurokinin receptor 1 (NK1R) are involved in macrophage mechano-transduction, similar to chondrocytes, and if alpha-calcitonin gene-related peptide (αCGRP) and the CGRP receptor (CRLR/Ramp1) show comparable activity. Murine RAW264.7 macrophages were subjected to a cyclic stretch for 1⁻3 days and 4 h/day. Loading and neuropeptide effects were analyzed for gene and protein expression of neuropeptides and their receptors, adhesion, apoptosis, proliferation and ROS activity. Murine bone marrow-derived macrophages (BMM) were isolated after surgical osteoarthritis (OA) induction and proliferation, apoptosis and osteoclastogenesis were analyzed in response to loading. Loading induced NK1R and CRLR/Ramp1 gene expression and altered protein expression in RAW264.7 macrophages. SP protein and mRNA level decreased after loading whereas αCGRP mRNA expression was stabilized. SP reduced adhesion in loaded RAW264.7 macrophages and both neuropeptides initially increased the ROS activity followed by a time-dependent suppression. OA induction sensitized BMM to caspase 3/7 mediated apoptosis after loading. Both sensory neuropeptides, SP and αCGRP, and their receptors are involved in murine macrophage mechano-transduction affecting neuropeptide impact on adhesion and ROS activity. OA induction altered BMM apoptosis in response to loading indicate that OA-associated biomechanical alterations might affect the macrophage population.