Pathologically Elevated-Cyclic Stretch Suppressed Vascular Smooth Muscle Cell Mitochondrial Biogenesis by Down-regulating PGC1α Expression / 医用生物力学

Objective To investigate the effect of pathologically elevated-cyclic stretch induced by hypertension on mitochondrial biogenesis of vascular smooth muscle cells (VSMCs), and the role of PGC1α in this process. Methods The Flexcell-5000T stretch loading system in vitro was applied to VSMCs with a frequency of 1. 25 Hz and an amplitude of 5% or 15% to simulate the mechanical environment under normal physiological or hypertensive pathological conditions respectively. Western blotting and qPCR were used to detect the expression of PGC1α, citrate synthase and mitochondrial DNA (mtDNA) copy number in VSMCs under normal physiological or hypertensive pathological conditions. VSMCs were treated with PGC1α specific activator ZLN005 to promote PGC1α expression or specific interfering fragment siRNA to inhibit PGC1α expression in order to detect the effect on citrate synthase and mtDNA copy number. Results Compared with 5% physiological cyclic stretch, 15% pathologically elevated-cyclic stretch significantly suppressed the expression of PGC1α, citrate synthase and mtDNA copy number in VSMCs. Compared with control group, the protein expression of PGC1α was significantly decreased and increased respectively. When VSMCs transfected with PGC1α siRNA or incubated PGC1α activator ZLN005, the expression of citrate synthase and mtDNA copy number were also significantly down regulated and up-regulated in VSMCs accordingly. Under physiological cyclic stretch conditions, the protein level of PGC1α was significantly down-regulated by PGC1α siRNA, which also significantly down-regulated citrate synthase expression and mtDNA copy number. The protein expression of PGC1α was significantly up-regulated by ZLN005, which also enhanced the expression of citrate synthase and mtDNA copy number. Conclusions The pathological cyclic stretch induced by hypertension significantly down-regulated the expression of citrate synthase and mtDNA copy number via suppressing the expression of PGC1α, resulting in mitochondrial dysfunction of VSMCs. PGC1α may be a potential therapeutic target molecule to alleviate the progression of hypertension.

Src/RUNX2 Modulates the Migration of Vascular Smooth Muscle Cells Induced by Cyclic Stretch / 医用生物力学

Objective To investigate the effect of cyclic stretch on Src and Runt-related transcription factor 2 (RUNX2), and their pivotal roles in migration of vascular smooth muscle cells (VSMCs). Methods The 5% cyclic stretch (to simulate normotensive physiological condition) or 15% cyclic stretch (to simulate hypertensive pathological condition) was applied to VSMCs by FX-5000T system. Western blotting was used to detect the expression of RUNX2 and phosphorylation of Src in VSMCs. The Ingenuity Pathway Analysis (IPA) bioinformatic software was used to analyze the potential regulatory effect of Src on RUNX2. Small interfering RNA (siRNA) was transfected to decrease the expression of RUNX2. Src inhibitor-1 was used to repress Src kinase activity; Wound-healing assay was applied to detect VSMC migration. Results Compared with 5% cyclic stretch, 15% cyclic stretch significantly increased RUNX2 expression in VSMCs. Under both static and 15% cyclic stretch conditions, VSMC migration was significantly inhibited after reducing RUNX2 expression with siRNA transfection. IPA indicated that Src kinase might be the upstream modulator of RUNX2, and Western blotting validated that RUNX2 expression was significantly decreased after inhibiting Src. Furthermore, under 15% cyclic stretch, Src inhibitor-1 markedly repressed RUNX2 expression and VSMC migration.Conclusions High cyclic stretch increased phosphorylation of Src kinase and expression of RUNX2, which subsequently induced VSMC abnormal migration. Exploring the mechanobiological mechanism of VSMC migration regulated by cyclic stretch may contribute to further revealing the mechanism of vascular physiological homeostasis and vascular pathological remodeling, as well as providing new perspective for the translational research of vascular remodeling upon hypertension.

Physiological Cyclic Stretch Inhibits Vascular Smooth Muscle Cell Migration via Activating AMPK Phosphorylation / 医用生物力学

Objective To explore the role of adenosine monophosphate-activated protein kinase (AMPK), a key regulator of cellular energy metabolism, in vascular smooth muscle cell (VSMC) migration in response to physiological cyclic stretch. Methods The Flexcell-5000T mechanical loading system was applied with a physiological cyclic stretch at 10% amplitude and 1.25 Hz frequency to primary rat VSMCs, to simulate mechanical stimulation of VSMCs in vivo. The protein expression of p-AMPK in VSMCs was detected by Western blotting, and VSMC migration was detected by wound healing test. Results Compared with the static group, physiological cyclic stretch loading for 24 h significantly decreased the area of wound healing, indicating that physiological cyclic stretch inhibited VSMC migration. The protein expression of p-AMPK in VSMCs was increased significantly after physiological cyclic stretch loading for 3 h, and was decreased significantly after 24 h. Under physiological cyclic stretch loading conditions, incubating AMPK inhibitor could significantly reduce the protein expression of p-AMPK after 3 h, and promote VSMC migration after 24 h; incubating AMPK activator AICAR under static conditions significantly increased the protein expression of p-AMPK after 3 h, and weakened VSMC migration after 24 h. Conclusions Physiological cyclic stretch inhibits VSMC migration by increasing the protein expression of p-AMPK, indicating that VSMC migration regulated by physiological cyclic stretch is of great significance for maintaining vascular homeostasis.

Calcium Participates in Migration of VSMCS Synergistically Induced by Cyclic Stretch and Platelet-Derived Microvesicles / 医用生物力学

Objective To investigate the synergistic effects of pathologically elevated cyclic stretch and platelet-derived microvesicles (PMVs) on migration of vascular smooth muscle cells (VSMCs) and the potential role of calcium in this process. Methods The FX-5000T strain loading system was used to apply cyclic stretch to VSMCs with magnitudes of 5% and 15%, which simulated physiological and hypertensive situation respectively in vitro; wound healing assay was used to analyze VSMCs migration; Ca2+-free medium was used to remove extracellular calcium; 2-APB (an antagonist of IP3R) was used to inhibit the release of intercellular stored calcium; GSK219 (an antagonist of TRPV4) and Nifedipine (an inhibitor of L-type voltage-gated calcium channel) were applied to block the activity of respective calcium channel; thrombin was used to stimulate platelets in vitro which simulated the hypertensive activation of PMVs in vivo. ResultsCompared with 5% cyclic stretch, 15% cyclic stretch significantly promoted VSMC migration. Removal of extracellular calcium inhibited VSMCs migration, but the application of GSK219 and Nifedipine did not affect the migration up-regulated by 15% cyclic stretch; while 2-APB which inhibited the release of intracellular stored calcium could also repress VSMCs migration under 15% cyclic stretch. PMVs further promoted VSMC migration under 15% cyclic stretch condition, and both extracellular calcium and intercellular stored calcium were involved in this process. Conclusions Both intracellular and extracellular calcium play important roles in VSMC migration induced by 15% cyclic stretch, and PMVs synergistically participate in the above process. The study is aimed to provide new mechanobiological insights into the molecular mechanism and clinical targets of vascular remodeling in hypertension.

Cyclic Stretch Induces Adhesion of VSMCs with Platelet-Derived Microparticles and the Role in Autophagy / 医用生物力学

Objective To investigate the effect of cyclic stretch on adhesion of vascular smooth muscle cells (VSMCs) with platelet-derived microparticles (PMPs), and the role of PMPs in VSMC autophagy. Methods Cyclic stretch with the magnitude of 5% (simulating physiological mechanical stretch) or 15% (simulating pathological mechanical stretch) was subjected to VSMCs in vitro by using FX-5000T cyclic stretch loading system, and the adhesion of PMPs in VSMCs was detected by using flow cytometry. Immunofluorescence was used to detect the expression of autophagy microtubule associated protein light chain 3 (LC3) after 24 h stimulation with PMPs. Western blotting was used to detect the expression of autophagy related protein (Atg) in VSMCs after 24 h stimulation by PMPs. Results Compared with 5% cyclic stretch, 15% cyclic stretch significantly increased the adhesion ability of VSMCs with PMPs. Immunofluorescence and Western blotting result revealed that PMPs stimulation significantly increased the expression of autophagy marker protein LC3 in VSMCs. Furthermore, the protein expressions of Atg5, Atg7 and Atg12 were all significantly increased in VSMCs stimulated with PMPs. Conclusions High cyclic stretch may enhance the autophagy of VSMCs by promoting the adhesion of PMPs, which will subsequently increase the expressions of Atg5, Atg7, Atg12 and LC3.

The Role of MicroRNA-214-3p in Differentiation and Proliferation of Endothelial Progenitor Cells in Response to Cyclic Stretch / 医用生物力学

Objective To investigate the role of microRNA-214-3p (miR-214-3p) in differentiation and proliferation of endothelial progenitor cells (EPCs) induced by cyclic stretch. Methods EPCs were exposed to cyclic stretch at physiological level (with the magnitude of 5%, at a constant frequency of 1.25 Hz) for 24 h by FX-5000T Strain Unit. miRNAs array was performed to identify the expression profiling of miRNAs. Real-time PCR was used to examine the expression levels of miRs. The expression of vascular smooth muscle cells (VSMCs) markers in EPCs was detected by real-time PCR. EPC proliferation was detected by BrdU ELISA assay. After EPCs were transfected with miR-214-3p inhibitor (IN) to knockdown expression of miR-214-3p, the level of VSMC markers expression and EPC proliferation was detected. Results Cyclic stretch significantly decreased miR-214-3p expression, depressed EPC differentiation toward VSMCs, and increased EPCs proliferation. Similarly, transfection with the miR-214-3p inhibitor led to the decreased expression of VSMC markers under static station. Meanwhile, miR-214-3p down-regulation promoted EPC proliferation significantly. Conclusions Physiological cyclic stretch could down-regulate the expression of miR-214-3p in EPCs, depress EPC differentiation towards VSMC and promote EPC proliferation eventually. Therefore, the research findings provide a potential therapeutic strategy for treating vessel injuries.

Physiological Cyclic Stretch Inhibits Vascular Smooth Muscle Cell Apoptosis via Inducing Expression of Nuclear Envelope Protein Emerin / 医用生物力学

Objective To investigate the mechanical response of Emerin, a nuclear envelope protein, and its role in apoptosis of vascular smooth muscle cells (VSMCs) during cyclic stretch, and the potential effect of transcriptional factors in this process. Methods Physiological cyclic stretch with the magnitude of 5% and frequency of 1.25 Hz was subjected to VSMCs in vitro by using FX-5000T cyclic stretch loading system. VSMCs cultured under the same conditions but without applying mechanical stretch were used as the static control. The apoptosis of VSMCs was detected by using Cleaved-caspase3 ELISA kit, and the expression of Emerin was revealed by using Western blotting. The effects of Emerin on activities of 345 kinds of transcriptional factors in VSMCs were demonstrated with Protein/DNA array after Emerin specific RNA interference (RNAi) under static condition, and the potential transcriptional factors involved in VSMC apoptosis were analyzed with Ingenurity Pathway Analysis (IPA) software. Furthermore, the binding abilities of Emerin to the motif of 2 kinds of apoptosis-related transcriptional factors were detected with chromatin immunoprecipitation (CHIP) and qPCR. ResultsCompared with the static control, the apoptosis of VSMCs was significantly decreased by 5% cyclic stretch, which suggested a protective effect of physiological cyclic stretch. The expressions of Emerin in VSMCs was remarkably increased with 5% cyclic stretch applied for 6 h, 12 h and 24 h. Specific RNAi under static condition decreased the expressions of Emerin but increased the apoptosis of VSMCs. Emerin siRNA transfection remarkably increased (more than 2 times) the activities of 10 transcriptional factors that participated in cellular apoptosis, i.e. CREB-BP1, p300, p55, MAX, NRF-1, STAT1, STAT3, TEF1, TR and BZP. CHIP-qPCR result revealed that the binding ability of Emerin to specific mofit of STAT1 or STAT3 was significantly repressed with Emerin RNAi. Conclusions Physiological cyclic stretch could increase the expression of Emerin which might modulate the binding of Emerin to motifs of apoptosis-related transcriptional factors such as STAT1 and STAT3, regulate the activities of these factors, and then subsequently repress the VSMC apoptosis. The investigation on mechanobiological mechanisms of VSMC apoptosis induced by cyclic stretch may contribute to further understanding the physiological and pathological mechanisms of vascular homeostasis and vascular remodeling.

17β-Estradiol Changes The Human Cerebral Endothelial Cell Proteome Upon Exposure To Cyclic Stretch / Jurnal Sains Kesihatan Malaysia

Hypertension and estrogen deficiency in women have been identified as significant risk factors for cerebrovasculardiseases. Hypertension causes excessive vascular stretch and contributes to the initiation of cellular injury in bloodvessels while estrogen has been demonstrated to exert beneficial protective effects on the vascular system. Although thespecific biological outcomes exerted by either excessive stretch or estrogen exposure are well established, the combinedbiochemical effects of both stimuli remain unclear. Therefore, this study was conducted for quantitative proteomics studyon human cerebral microvascular endothelial cells (HCMECs) subjected to 20% “pathological” cyclic stretch for aperiod of 18 hour in the presence or absence of 17β-estradiol by isobaric Taqs for Relative and Absolute Quantification.The results showed that only some proteins responded to 17β-estradiol (e.g., thioredoxin reductase-1), stretch (e.g., 14-3-3 protein epsilon or acidic leucine-rich nuclear phosphoprotein 32 family member B) and interestingly, some proteinsreturned to control pre-treatment levels when exposed to both (e.g., d-dopachrome decarboxylase, thrombospondin-1). Inaddition, HCMECs that exposed only to estrogen had a very similar proteomic profile (i.e., up-regulation of structural,cellular adhesion and proliferation proteins) as to those exposed to estrogen with 20% stretching for 18 hour, suggestingthat estrogen ablated the detrimental effects by the stretch alone. These findings sheds light on the molecular mechanismsby which the cerebrovascular protective actions of estrogen on HCMEC exposed to pathological levels of cyclic stretchwhich could provide a platform for future research in therapeutic approach.

Stretch-induced Expression of CYR61 Increases the Secretion of IL-8 in A549 Cells via the NF-κβ/Iκβ Pathway / 华中科技大学学报（医学）（英德文版）

Mechanical ventilation (MV) with large tidal volumes can increase lung alveolar permeability and initiate inflammatory responses,resulting in ventilator-induced lung injury (VILI).The mechanisms of the injurious effects of MV and the genetic susceptibility remain unclear.VILI-related genes such as cysteine-rich angiogenic inducer 61 (Cyr61)have been demonstrated to play a detrimental role in the aggressive ventilation strategies.In the present study,we investigated the involvement of Cyr61 in the VIM and the underlying mechanism.A549 cells were exposed to cyclic stretch of varying durations and then the mRNA and protein levels of Cyr61 were measured by real-time PCR and Western blotting,respectively.Additionally,after exposure ofA549 cells to cyclic stretch for 5 min to 1 h,the expression levels of nuclear factor kappaB (NF-κB) and IL-8 were detected by ELISA and Western blotting.Thereafter,Cyr61 expression was depressed in A549 cells with the siRNA pGenesill.1-Cyr61-3 before the cyclic stretch,and IL-8 secretion and the activation of NF-κB pathways were probed by ELISA and Western blotting,respectively.Moreover,a NF-κB inhibitor (PDTC) and an activator (TNF) were used before mechanical stretch.Realtime PCR and ELISA were performed to detect the mRNA and protein of IL-8,respectively.The results showed that the mechanical cyclic stretch led to increased Cyr61 expression at mRNA and protein levels in A549 cells.Additionally,cyclic stretch also mobilized NF-κB from the cytoplasm to the nucleus and increased IL-8 secretion in A549 cells.The inhibition of Cyr61 blocked the NF-κB activation and IL-8 secretion in response to cyclic stretch.Inhibition of NF-κB attenuated the mRNA and protein expression of IL-8 in A549 cells transfected with Cyr61 siRNA.It was suggested that Cyr61/NF-κB signaling pathway mediates the upregulation of IL-8 in response to cyclic stretch in A594 cells.These findings support the hypothesis that Cyr61 plays a critical role in acute lung inflammation triggered by mechanical strain.

Stretch-induced Expression of CYR61 Increases the Secretion of IL-8 in A549 Cells via the NF-κβ/Iκβ Pathway / 华中科技大学学报（医学）（英德文版）

Mechanical ventilation (MV) with large tidal volumes can increase lung alveolar permeability and initiate inflammatory responses,resulting in ventilator-induced lung injury (VILI).The mechanisms of the injurious effects of MV and the genetic susceptibility remain unclear.VILI-related genes such as cysteine-rich angiogenic inducer 61 (Cyr61)have been demonstrated to play a detrimental role in the aggressive ventilation strategies.In the present study,we investigated the involvement of Cyr61 in the VIM and the underlying mechanism.A549 cells were exposed to cyclic stretch of varying durations and then the mRNA and protein levels of Cyr61 were measured by real-time PCR and Western blotting,respectively.Additionally,after exposure ofA549 cells to cyclic stretch for 5 min to 1 h,the expression levels of nuclear factor kappaB (NF-κB) and IL-8 were detected by ELISA and Western blotting.Thereafter,Cyr61 expression was depressed in A549 cells with the siRNA pGenesill.1-Cyr61-3 before the cyclic stretch,and IL-8 secretion and the activation of NF-κB pathways were probed by ELISA and Western blotting,respectively.Moreover,a NF-κB inhibitor (PDTC) and an activator (TNF) were used before mechanical stretch.Realtime PCR and ELISA were performed to detect the mRNA and protein of IL-8,respectively.The results showed that the mechanical cyclic stretch led to increased Cyr61 expression at mRNA and protein levels in A549 cells.Additionally,cyclic stretch also mobilized NF-κB from the cytoplasm to the nucleus and increased IL-8 secretion in A549 cells.The inhibition of Cyr61 blocked the NF-κB activation and IL-8 secretion in response to cyclic stretch.Inhibition of NF-κB attenuated the mRNA and protein expression of IL-8 in A549 cells transfected with Cyr61 siRNA.It was suggested that Cyr61/NF-κB signaling pathway mediates the upregulation of IL-8 in response to cyclic stretch in A594 cells.These findings support the hypothesis that Cyr61 plays a critical role in acute lung inflammation triggered by mechanical strain.

The role of extracellular signal regulated kinase 1/2 in mediating osteodifferentiation of human periodontal ligament cells induced by cyclic stretch / 华西口腔医学杂志

<p><b>OBJECTIVE</b>This study aimed to investigate the mechanism of cyclic stretch that promotesthe osteogenic differentiation of human periodontal ligament cells (hPDLCs) through the mediation of extracellular-signal-regulated kinase 1/2 (ERK1/2).</p><p><b>METHODS</b>hPDLCs were isolated through the explant method and cultured in vitro. hPDLCs were mechanically stimulated by a multi-channel cell-stress-loading system for 1, 3, 6, 12, and 24 h. The magnitude of stretch was 10% deformation, and the frequency was 0.5 Hz. Nonloaded cells were used as control group. ERK1/2 activation was blocked by U0126, a specific ERK1/2 pathway inhibitor. Additionally, hPDLCs were transfected with adenoviral vector encoding dominant negative ERK1/2 (DN-ERK1/2) to continuouslyinhibit ERK1/2 activation. The mRNA and protein levels of target geneswere detected through real-time polymerase chain reaction and Western blot.</p><p><b>RESULTS</b>Cyclic stretching promoted the expression of ERK1/2, osteocalcin (OCN) mRNA, and bone sialoprotein (BSP) mRNA. The expression of runt-related transcription factor (Runx) 2 protein and mRNA also increased at 3 and 6 h of cyclic stretching. The inhibition of ERK1/2 by U0126 and DN-ERK1/2 suppressed the expressionof Runx2 mRNA, OCN mRNA, BSP mRNA, Runx 2 protein, and p-ERK1/2 protein relative to that in stretched cells without the ERK1/2 inhibitor.</p><p><b>CONCLUSIONS</b>ERK1/2 is a critical molecule in the mediation ofthe osteogenic differentiation of hPDLCs under mechanical stimulation. ERK1/2 activation induced the elevation of Runx2 protein levels, which may be involved in the stretch-induced expressions of OCN and BSP.</p>

Cyclic Stretch Effects on Adipose-Derived Stem Cell Stiffness, Morphology and Smooth Muscle Cell Gene Expression

Recent investigations consider adipose-derived stemcells (ASCs) as a promising source of stemcells for clinical therapies. To obtain functional cells with enhanced cytoskeleton and aligned structure, mechanical stimuli are utilized during differentiation of stem cells to the target cells. Since function of muscle cells is associated with cytoskeleton, enhanced structure is especially essential for these cells when employed in tissue engineering. In this study by utilizing a custom-made device, effects of uniaxial tension (1Hz, 10% stretch) on cytoskeleton, cell alignment, cell elastic properties, and expression of smooth muscle cell (SMC) genes in ASCs are investigated.Due to proper availability ofASCs, results can be employed in cardiovascular engineeringwhen production of functional SMCs in arterial reconstruction is required. Results demonstrated that cells were oriented after 24 hours of cyclic stretch with aligned pseudo-podia. Staining of actin filaments confirmed enhanced polymerization and alignment of stress fibers. Such phenomenon resulted in stiffening of cell body which was quantified by atomic force microscopy (AFM). Expression of SM α-actin and SM22 α-actin as SMC associated genes were increased after cyclic stretch while GAPDH was considered as internal control gene. Finally, it was concluded that application of cyclic stretch on ASCs assists differentiation to SMC and enhances functionality of cells.

Effects of mechanical stretch on gene expression of extracellular matrix in rabbit corneal fibroblasts / 医用生物力学

Objective To explore the combined effects of mechanical stretch and interleukin-1β (IL-1β) on gene expression of extracellular matrix in rabbit corneal fibroblasts. Methods Isolated rabbit corneal fibroblasts were subjected to 15% equibiaxial stretch at frequency of 0.1 Hz for 12 h, 24 h and 36 h, respectively, in presence of IL-1β. The gene expressions of matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinases 1 (TIMP-1) and collagen type I alpha 1 (Collagen Iα1) were detected by real-time quantitative PCR. Results The mRNA levels of MMP-1, MMP-3 and MMP-9 could be up-regulated by IL-1β alone. However, MMP-1 and MMP-3 mRNA levels decreased with time, while MMP-9, TIMP-1 and collagen Iα1 increased with time. Compared with corresponding IL-1β treatment with mechanical stretch groups, the mRNA levels of MMP-1, MMP-3 and MMP-9 were increased and the mRNA levels of TIMP-1 and collagen Iα1 were decreased in a time-dependent manner. The mRNA level of Collagen Iα1 was decreased by loading mechanical stretch alone, and would further decrease time-dependently in combination with IL-1β treatment. Conclusions Mechanical stretch combined with IL-1β may facilitate the corneal tissue damage, thereby contribute to the development of keratectasia.

The role of FOXO1 in cyclic stretch induced-proliferation of vascular smooth muscle cells during hypertension / 医用生物力学

Objective To investigate the role of pathologically increased-cyclic stretch in proliferation of vascular smooth muscle cells (VSMCs) during hypertension, and the effect of Forkhead box protein O1 (FOXO1) during this process. Methods Coarctation of abdominal aorta above kidney artery of rat was used as hypertensive animal model, and sham-operated animal as control. FX-4000 cyclic stretch loading system was used to apply 5% physiologically cyclic stretch and 15% pathologically cyclic stretch during hypertension on VSMCs in vitro. Western blot was used to reveal the expressions of FOXO1 and phosphor-FOXO1 in VSMCs, and BrdU kit to detect the proliferation of VSMCs in vitro. By using RNA interference in static, the role of FOXO1 on cell proliferation was further detected. Results After abdominal aorta coarctation for 2 and 4 weeks, respectively, the blood pressure was significantly increased compared with the sham operated rats. The proliferation of vascular cells in aorta of hypertensive rat was significantly increased, and so did the expressions of FOXO1 and phosphor-FOXO1. In vitro experiment revealed that 15% cyclic stretch remarkably increased the proliferation and expressions of FOXO1 and phospho FOXO1 in VSMCs. Target siRNA transfection in static decreased the expression of FOXO1 and phosphor-FOXO1, as well as the proliferation of VSMCs. Conclusions Pathologically increased-cyclic stretch may increase the expression and phosphorylation of FOXO1, subsequently modulate VSMC proliferation during hypertension. Based on animal models, this study intends to reveal the role of FOXO1 in vascular reconstruction of hypertension and the involved biomechanical mechanism, so as to make the mechanobiological mechanism of hypertension explicit and discover new target in the prevention and treatment of vascular remodeling.

Cyclic stretch-induced apoptosis in human osteoarthritic chondrocytes is mediated by reactive oxygen species generation / 医用生物力学

Objective To explore the mechanism of reactive oxygen species (ROS) generation on apoptosis of human osteoarthritic chondrocytes induced by cyclic stretch in vitro. Methods The human osteoarthritic chondrocytes were subjected to cyclic stretch at the frequency of 0.5 Hz with 20% elongation. The chondrocytes without cyclic stretch were used as a control. ROS generation in chondrocytes was inhibited by the antioxidant, N-acetyl-L-cysteine (NAC) and potentiated by the glutathione depleter, DL-buthionine-［S,R］-sulfoximine (BSO). Apoptosis was detected by flow cytometry. Intracellular ROS was detected using DCFH-DA and caspase-9 activity was measured using spectrophotometry. Results The cyclic stretch at the frequency of 0.5 Hz with 20% elongation induced ROS generation, and activation of caspase-9 and apoptosis in human osteoarthritic chondrocytes were significantly increased (P<0.05). The inhibition or potentiation of intracellular ROS by NAC or BSO could obviously inhibit or improve caspase-9 activity and apoptosis in chondrocytes under cyclic stretch (P<0.05). Conclusions Cyclic stretch-induced apoptosis in human osteoarthritic chondrocytes is mediated by ROS generation and activation of caspase-9. Suppression of ROS can prevent chondrocytes from apoptosis induced by cyclic stretch.

Effects of mechanical stimulation on basic fibroblast growth factor expression of corneal fibroblasts / 医用生物力学

Objective To investigate the effect of different mechanical environment ( in vivo and in vitro) on expression of basic fibroblast growth factor (bFGF) and explore the role of mechanical stimulation in corneal tissue repair after laser assisted in situ keratomileusis (LASIK) surgery. Methods Animal models by LASIK surgery were established to keep the corneas under different mechanical environment. The experimental animals were killed at the first week or the first month after LASIK surgery to obtain the corneas. In addition, the primary corneal fibroblasts were subjected to cyclic mechanical stretch (0.1 Hz; 5%, 10%, 15% stretch; 6 h or 24 h) using Flexcell 4 000 tension system. Expression of bFGF was determined by ELISA method. Results At the first week after LASIK surgery, expression of bFGF was increased significantly in 30% group (residual stroma bed accounting for 30% of the whole cornea), as compared with the control group (P<0.05), and then it was decreased to the normal level in all groups at the first month after LASIK surgery. Analysis on the same surgery method at different time showed that there were significant differences only in 30% group at the first week and month (P<0.05). Cyclic stretch experiment in vitro indicated that bFGF expression in 15% stretch group was significantly increased after 6 h than that in the control group (P<0.05), with a significant decrease after 24 h (P<0.05). Conclusions Mechanical stimulation can regulate bFGF expression of corneal tissues and corneal frbroblasts, and bFGF plays a positive role in the early corneal tissue repair after LASIK surgery.

Effects of cyclic mechanical stretch on BMP-2 expression in RA and OA fibroblast-like synoviocytes / 医用生物力学

Objective To explore the role of mechanical stimulation in synovium under different pathological conditions through studying the effects of cyclic mechanical stretch on the expression of bone morphogenetic protein 2 (BMP-2) in rheumatoid arthritis (RA) and osteoarthritis (OA) fibroblast-like synoviocytes (FLS). Method 6% and 0.5 Hz stretch generated by Flex cell 4000 tension systems was applied on normal, RA and OA FLS of human knee joint source under normal and inflammatory conditions for 2 h or 6 h, respectively. Results Cyclic mechanical stretch of 6%, 0.5 Hz had no significant effects on the expression of BMP-2 in normal, RA and OA FLS at 2 h, while in RA FLS it increased significantly at 6 h. Inflammatory cytokines (IL-1β) didn’t influence normal FLS at 2 h, but made BMP-2 mRNA significantly increased at 6 h. IL-1β increased BMP-2 mRNA of RA FLS significantly both at 2 h and 6 h. IL-1β increased BMP-2 mRNA of OA FLS significantly only at 2 h, but had no significant effect at 6 h. The co-effect of IL-1β and cyclic mechanical stretch induced the ascension of BMP-2 expression significantly in normal and RA FLS at 2 h, and in normal, RA and OA FLS at 6 h. Conclusions Response of BMP-2 mRNA to mechanic stimulation and IL-1β in normal, RA and OA FLS were different. Inflammation may play a more important role than mechanical stimulation in the pathogenesis of RA and OA. Synergetic effect in inflammation and mechanical stimulation were found in OA FLS at 6 h, which reveals that they may co-act in the occurrence and development of OA.

Role of Ca~(2+) signaling in overexpression of detrusor smooth muscle cell connexin 43 by cyclic stretch in vitro / 第三军医大学学报

Objective To investigate the role of Ca 2+ signaling in the overexpression of detrusor smooth muscle cell connexin 43 by cyclic stretch in vitro. Methods The detrusor smooth muscle cells (DSMC) grown on collagen-coated silicone membranes were subjected to cyclic stretch-relaxation. The Cx43 mRNA in DSMC were detected with RT-PCR, and the concentration of intracellular free Ca 2+ in DSMC was measured by confocal microscopy in conjunction with the calcium indicator, Fura-3 (Molecular Probes). Results The overexpression of Cx43 mRNA induced by cyclic stretch was significantly inhibited by EGTA.The increase of intracellular free Ca 2+ induced by stretch was also inhibited completely by EGTA, 61.95% by GdCl3, 29.98% by Nifedipine, 87.98% by Ryanodine and Thapsigargin. Conclusion The stretch-induced Ca 2+ entry, via the Ca 2+-induced Ca 2+ release mechanism, may play an important role in DSMC connexin 43 overexpression induced by cyclic stretch.