Effects of Cyclic Strain on Neural Differentiation of Rat Bone Marrow-Derived Mesenchymal Stem Cells / 医用生物力学

Objective To study the mechanical effects of cyclic strain on neural differentiation of rat bone marrow mesenchymal stem cells (rBMSCs). Methods The rBMSCs were subjected to cyclic strain for 24 hours andthen cultured for 5 days. The expression of neural markers and the phosphorylation of relative signaling pathway proteins were evaluated. The stress distribution on cell surface was analyzed by finite element method. The differentially expressed genes induced by strain were identified by RNA sequencing analysis. Results The 0. 5 Hz strain with 5% magnitude could significantly induce higher expression of neural markers and elevated phosphorylation level of extracellular-signal-regulated kinase (ERK), protein kinase B (AKT) and mammalian target of rapamycin ( mTOR). KEGG pathway analysis showed that the focal adhesion and ECM-receptor interaction were significantly enriched under cyclic strain. Conclusions Cyclic strain could change the interaction of cells with the extracellular matrix ( ECM) and enhance the AKT/ mTOR and ERK pathway, finally promote rBMSC neural differentiation. Knowledge about the impact of mechanical stimulation on BMSC neural differentiation is expected to improve the efficiency of stem cell differentiation, shed light on device design for tissue engineering, and promote clinical application of mesenchymal stem cells in neural issue repair and regeneration.

ROCK1 and the relative signal molecules participate in proliferation of vascular smooth muscle cells induced by cyclic strain / 医用生物力学

Objective To investigate the role of Rho-associated coiled-coil containing protein kinase 1 (ROCK1) and the relative signal molecules in sensing the mechanical stimulation from tensile strain and regulating the proliferation of vascular smooth muscle cells (VSMCs).Methods Physiological cyclic strain with magnitude of 10％ and at frequency of 1.25 Hz was applied to VSMCs in vitro by using the strain loading system.The proliferation level of VSMCs was analyzed by BrdU ELISA;the expression level of ROCK1,phosphorylations of protein kinase C (PKC) α/β Ⅱ,protein kinase D (PKD) and extracellular regulated protein kinase (ERK) in VSMCs modulated by cyclic strain were detected with Western blotting;the expression of ROCK1 was specifically repressed by using RNA interference (RNAi).Results Compared with the static control,10％ cyclic strain significantly decreased the expression of ROCK1 and phosphorylations of PKD and ERK.The phosphorylation of PKCα/βⅡ decreased significantly under 10％ cyclic strain for 12 h,but returned to normal level after loading for 24 h.Repressed expression of ROCK1 with RNAi significantly down-regulated VSMC proliferation,suppressed phosphorylations of PKCα/βⅡ and PKD,but no obvious changes were found in phosphorylation of ERK.Conclusions Physiological cyclic strain with magnitude of 10％ may repress the phosphorylation of PKCα/βⅡ and PKD via inhibiting the expression of ROCK1,and subsequently affects VSMC proliferation and maintains vascular hemostasis.The investigation on intracellular mechanotransduction network of VSMCs under mechanical stimulation of cyclic strain may contribute to studying the physiological and pathological mechanisms of cardiovascular diseases.

ROCK1 and the relative signal molecules participate in proliferation of vascular smooth muscle cells induced by cyclic strain / 医用生物力学

Objective To investigate the role of rho-associated coiled-coil containing protein kinase 1 (ROCK1) and the relative signal molecules in sensing the mechanical stimulation from tensile strain and regulating the proliferation of vascular smooth muscle cells (VSMCs). Methods Physiological cyclic strain with magnitude of 10% and at frequency of 1.25 Hz was applied to VSMCs in vitro by using strain loading system. The proliferation level of VSMCs was analyzed by BrdU ELISA; the expression level of ROCK1, phosphorylations of protein kinase C (PKC) α/β II, protein kinase D (PKD) and extracellular regulated protein kinase (ERK) in VSMCs modulated by cyclic strain were detected with Western blotting; the expression of ROCK1 was specifically repressed by using RNA interference (RNAi). Results Compared with the static control, 10% cyclic strain significantly decreased the expression of ROCK1 and phosphorylations of PKD and ERK. The phosphorylation of PKCα/βII was decreased significantly under 10% cyclic strain for 12 h, but returned to normal level after 24 h-loading. Repressed expression of ROCK1 with RNAi significantly down-regulated VSMC proliferation, suppressed phosphorylations of PKCα/βII and PKD, but no obvious change was found in phosphorylation of ERK. Conclusions Physiological cyclic strain with magnitude of 10% may repress the phosphorylation of PKCα/βII and PKD via inhibiting the expression of ROCK1, which subsequently affect VSMC proliferation and maintain vascular hemostasis. The investigation on intracellular mechanotransduction network of VSMCs under mechanical stimulation of cyclic strain may contribute to the physiological and pathological mechanisms of cardiovascular diseases.

ROCK1 and the relative signal molecules participate in proliferation of vascular smooth muscle cells induced by cyclic strain / 医用生物力学

Objective To investigate the role of Rho-associated coiled-coil containing protein kinase 1 (ROCK1) and the relative signal molecules in sensing the mechanical stimulation from tensile strain and regulating the proliferation of vascular smooth muscle cells (VSMCs).Methods Physiological cyclic strain with magnitude of 10％ and at frequency of 1.25 Hz was applied to VSMCs in vitro by using the strain loading system.The proliferation level of VSMCs was analyzed by BrdU ELISA;the expression level of ROCK1,phosphorylations of protein kinase C (PKC) α/β Ⅱ,protein kinase D (PKD) and extracellular regulated protein kinase (ERK) in VSMCs modulated by cyclic strain were detected with Western blotting;the expression of ROCK1 was specifically repressed by using RNA interference (RNAi).Results Compared with the static control,10％ cyclic strain significantly decreased the expression of ROCK1 and phosphorylations of PKD and ERK.The phosphorylation of PKCα/βⅡ decreased significantly under 10％ cyclic strain for 12 h,but returned to normal level after loading for 24 h.Repressed expression of ROCK1 with RNAi significantly down-regulated VSMC proliferation,suppressed phosphorylations of PKCα/βⅡ and PKD,but no obvious changes were found in phosphorylation of ERK.Conclusions Physiological cyclic strain with magnitude of 10％ may repress the phosphorylation of PKCα/βⅡ and PKD via inhibiting the expression of ROCK1,and subsequently affects VSMC proliferation and maintains vascular hemostasis.The investigation on intracellular mechanotransduction network of VSMCs under mechanical stimulation of cyclic strain may contribute to studying the physiological and pathological mechanisms of cardiovascular diseases.

ROCK1 and the relative signal molecules participate in proliferation of vascular smooth muscle cells induced by cyclic strain / 医用生物力学

Objective To investigate the role of Rho-associated coiled-coil containing protein kinase 1 (ROCK1) and the relative signal molecules in sensing the mechanical stimulation from tensile strain and regulating the proliferation of vascular smooth muscle cells (VSMCs).Methods Physiological cyclic strain with magnitude of 10％ and at frequency of 1.25 Hz was applied to VSMCs in vitro by using the strain loading system.The proliferation level of VSMCs was analyzed by BrdU ELISA;the expression level of ROCK1,phosphorylations of protein kinase C (PKC) α/β Ⅱ,protein kinase D (PKD) and extracellular regulated protein kinase (ERK) in VSMCs modulated by cyclic strain were detected with Western blotting;the expression of ROCK1 was specifically repressed by using RNA interference (RNAi).Results Compared with the static control,10％ cyclic strain significantly decreased the expression of ROCK1 and phosphorylations of PKD and ERK.The phosphorylation of PKCα/βⅡ decreased significantly under 10％ cyclic strain for 12 h,but returned to normal level after loading for 24 h.Repressed expression of ROCK1 with RNAi significantly down-regulated VSMC proliferation,suppressed phosphorylations of PKCα/βⅡ and PKD,but no obvious changes were found in phosphorylation of ERK.Conclusions Physiological cyclic strain with magnitude of 10％ may repress the phosphorylation of PKCα/βⅡ and PKD via inhibiting the expression of ROCK1,and subsequently affects VSMC proliferation and maintains vascular hemostasis.The investigation on intracellular mechanotransduction network of VSMCs under mechanical stimulation of cyclic strain may contribute to studying the physiological and pathological mechanisms of cardiovascular diseases.

The role of lncRNA-XR007793 in hypertensively cyclic strain induced-proliferation of vascular smooth muscle cells / 医用生物力学

Objective To investigate the effect of pathologically elevated cyclic strain induced by hypertension on proliferation of vascular smooth muscle cells (VSMCs) and the role of long non-coding RNA (IncRNA)-XR007793 during this process. Methods Flexcell-4000 tension system was used to apply physiologically (5% magnitude) and pathologically (15% magnitude) cyclic strain with frequency of 1.25 Hz on VSMCs for 24 h respectively. qRT-PCR was used to detect the expression of XR007793 and 4 co-expressed genes: signal transducer and activator of transcription 2 (STAT2), cell division cycle associated 8 (CDCA8), proto-oncogene LMO2 and interferon regulatory factor (IRF7). Western blot was used to detect the proliferating cell nuclear antigen (PCNA) level in VSMCs. RNA inference was used to inhibit XR007793 expression. The cell cycle of VSMCs was measured by flow cytometry in static condition and the cell proliferation was detected by Brdu-Elisa in cyclic strain loading condition. Results Compared with 5% cyclic strain, 15% cyclic strain remarkably decreased the XR007793 level and increased the proliferation of VSMCs，along with the increasing expression of STAT2 and CDCA8. XR007793 specific siRNA transfection under static condition decreased the expression of XR007793 and increased the VSMC proliferation. Under 15% cyclic strain, XR007793 specific siRNA transfection also increased the VSMC proliferation and the expression of CDCA8 compared with the non-specific siRNA control. Conclusions Pathologically elevated cyclic strain decreases the XR007793 expression level and increases the CDCA8 expression level to modulate VSMC proliferation. These results provide new experimental evidence for the study of mechanobiological mechanism during hypertension and potential targets for hypertension therapy.

TNF-α promotes the quantity and ICAM-1 expression of endothelial microparticles / 医用生物力学

Objective To study the role of cyclic strain-modulated tumor necrosis factor-α (TNF-α) played in the quantity and intercellular cell adhesion molecule-1(ICAM-1) expression of endothelial microparticles (EMPs). Methods The endothelial cells (ECs) primarily cultured from rat aorta were applied with 5% cyclic strain (to simulate normal physiological condition) and 18% cyclic strain (to simulate hyper-tension condition), respectively, by using FX-4000T cyclic stain loading system for 24 hours at the loading frequency of 1.25 Hz. The mRNA expression of TNF-α under different amplitudes of cyclic strain was determined by real time-PCR. The TNF-α was then used to stimulate the ECs from rat aorta, and the supernatants were collected and ultracentrifuged to get endothelial microparticles (EMPs), which were then identified by lipophilic styryl membrane staining and transmission electron microscope for morphological identification. The quantities of Annexin V positive EMPs under TNF-α stimulation were counted by flow cytometer and ICAM-1 expression on EMPs was detected as well. Results Compared with the 5% normal cyclic strain, under 18% high cyclic strain condition,the mRNA expression of TNF-α in ECs increased significantly. TNF-α could then significantly up-regulate the production of Annexin V positive EMPs and promote the expression of ICAM-1 on EMPs. Conclusions The over-expression of TNF-α in ECs under high cyclic strain might mediate the high production of EMPs and over-expression of ICAM-1 on EMPs. The research findings will provide new experiment evidence for further studying the role of EPCs in the mechanobiological mechanism of vascular remodeling.

Regulating effect of microRNA-21 on the expression of extracellular matrix of vascular smooth muscle cells under vascular remodeling of hypertension / 医用生物力学

Objective To investigate the regulating effect and mechanism of microRNA-21 (miR-21) on extracellular matrix (ECM) of vascular smooth muscle cells (VSMCs) by vascular remodeling of hypertension. Methods By narrowing the abdominal aorta in rats, the hypertension models were established and divided into 2-week hypertension group and 4-week hypertension group, and sham-operated group was also established as control. VSMCs from the rat aorta were subjected to 0% (static), 5% (normal) and 15%（hypertensive）elongation strain at a constant frequency of 1.25 Hz and duration of 12 hours, respectively. The expressions of Smad 7 and ECM were detected by Western blotting, and the expression of miR-21 was examined by Real-time RT-PCR. Finally, miR-21 siRNA was used to study the role of miR-21 in the mechanical strain-induced expression of ECM, miR-21 and Smad 7. Results Compared with the sham-operated group, ECM and miR-21 in thoracic aorta of 2-week hypertension group were significantly elevated. Collagen I, collagen III and miR-21 in thoracic aorta of 4-week hypertension group were significantly elevated. Compared with the static and 5% strain groups, the protein expression of collagen I in VSMCs did not show significant change, but the protein expression of collagen III was significantly elevated and Smad 7 expression was significantly decreased in 15% strain group. The cyclic strain also enhanced miR-21 expression in VSMCs. miR-21 inhibitor effectively decreased the expression of miR-21 in VSMCs and protein level of collagen III, while enhanced Smad 7 expression under the static and 15% strain. Conclusions The vascular remodeling of hypertension causes the high expressions of ECM and miR-21. The cyclic strain induces the high expression of miR-21, which via Smad 7 results in enhancing the expression of ECM, collagen III especially, in VSMCs under vascular remodeling of hypertension.

Application of nonlinear model in analysis on viscoelastic properties of the intervertebral disc / 医用生物力学

Objective To construct a variable-parameter nonlinear model for the research on stress relaxation properties of human intervertebral disc under the cyclic strain. Methods The variable-parameter nonlinear model combined with experimental data on stress relaxation and creep response of the intervertebral disc were used to study stress relaxation properties under the cyclic strain and compare the differences of linear and nonlinear model in viscoelastic properties of the intervertebral disc. Results The cycle modulus and relaxation coefficient obtained by the variable-parameter nonlinear model under the of frequency 0.01 Hz was very close to the experimental data, and the cyclic modulus under the frequency of 0.1 and 1 Hz were also close to the experimental data, but the relaxation coefficient obtained in 0.1 and 1 Hz had serious distortion. Conclusions The intervertebral disc experiences a nonlinear stress behavior under the compression strain, so the variable-parameter nonlinear model is more suitable for studying the stress relaxation response of the intervertebral disc under the cyclic strain.

Effects of combined mechanical stimulation on the proliferation and differentiation of pre-osteoblasts

We observed how combined mechanical stimuli affect the proliferation and differentiation of pre-osteoblasts. For this research, a bioreactor system was developed that can simultaneously stimulate cells with cyclic strain and ultrasound, each of which is known to effectively stimulate bone tissue regeneration. MC3T3-E1 pre-osteoblasts were chosen for bone tissue engineering due to their osteoblast-like characteristics. 3-D scaffolds were fabricated with polycaprolactone and poly-L-lactic acid using the salt leaching method. The cells were stimulated by the bioreactor with cyclic strain and ultrasound. The bioreactor was set at a frequency of 1.0 Hz and 10% strain for cyclic strain and 1.0 MHz and 30 mW/cm2 for ultrasound. Three experimental groups (ultrasound, cyclic strain, and combined stimulation) and a control group were examined. Each group was stimulated for 20 min/day. Mechanical stimuli did not affect MC3T3-E1 cell proliferation significantly up to 10 days when measured with the cell counting kit-8. However, gene expression analysis of collagen type-I, osteocalcin, RUNX2, and osterix revealed that the combined mechanical stimulation accelerated the matrix maturation of MC3T3-E1 cells. These results indicate that the combined mechanical stimulation can enhance the differentiation of pre-osteoblasts more efficiently than simple stimuli, in spite of no effect on cell proliferation.

The Difference of Akt Phosphorylation with Different Strengths of Hemodynamic Forces on the Endothelial Cell

PURPOSE: Mechanical forces including shear stress (SS) and cyclic strain (CS) trigger signal transducing events and modulate gene expression in vascular endothelial cells (ECs). However, differences in intracellular events and gene expression depend on the intensity of the forces, which remain unknown. We attempted to clarify the effect of different strengths of SS and CS by determination of the Akt phosphorylation of ECs. METHOD: ECs were exposed to the arterial level of orbital SS with an orbital shaker (210 rpm) or laminar SS (14 dyne/cm2) with a parallel plate or CS (10%) at a frequency of 60 cycles/min. Akt phosphorylation was assessed by immunocytochemical staining and Western blotting with specific antibodies against the phosphorylated Akt. RESULT: Akt demonstrated a time-dependent stimulation of Akt phosphorylation by mechanical forces with a maximal increase up to 2.5-fold after 30 minutes of orbital SS exposure, 3-fold after 2 minutes of laminar SS exposure and 2.3-fold after 30 minutes of CS exposure. Akt phosphorylation in the periphery of the membrane was significantly increased, compared to at the center of the membrane. It was up to 1.4-fold after 30 minutes of orbital SS and up to 2.4-fold after 30 minutes of CS (P<0.05). CONCLUSION: Our results demonstrate that all types of hemodynamic forces phosphorylated Akt in a time-dependent manner and different types of SS may have different effects on the ECs. In addition, the phosphorylation of Akt is dependent on the intensity of the mechanical forces on ECs.

The Role of PKCzeta on MT1-MMP Expression with Shear Stress and Cyclic Strain in Microvascular Endothelial Cells

PURPOSE: hear stress (SS) and cyclic strain (CS) influence the expression of membrane type 1-matrix metalloproteinase (MT1-MMP) in microvascular endothelial cells (MVECs). It is known that changes in the level of Sp1 phosphorylation are important for MT1-MMP expression following SS and CS. However, the exact mechanism underlying this process is poorly understood. The aim of this study was to determine the effect of PKCzeta on serine phosphorylation and activation of Sp1 in response to SS and CS. METHOD: MVECs were exposed to SS or CS for up to 8 hours with or without PKCzeta inhibitors. The activity and phosphorylation of Sp1 were assessed by Western blot analysis and immunoprecipitation. MT1-MMP protein expression was assessed by Western blot analysis. RESULT: PKCzeta was phosphorylated and activated under SS, whereas no significant changes were noted under CS. SS increased Sp1 phosphorylation in a time-dependent manner, but no changes in the Sp1 phosphorylation were observed when the MVECs were pretreated with the PKCzeta inhibitors. By contrast, MVECs exposed to CS in the presence or absence of PKCzeta inhibitors showed no change in the phosphorylation of Sp1. SS decreased MT1-MMP protein expression in a time-dependent manner, but in the presence of PKCzeta inhibitors, MT1-MMP expression was not changed compared with the static levels after SS. CS increases MT1-MMP expression in a time-dependent manner. Similar expression was observed when the cells were pretreated with PKCzeta inhibitors under CS. CONCLUSION: These data demonstrate that the increased affinity of Sp1 for the MT1-MMP's promoter site occurs because of PKCzeta induced phosphorylation of Sp1 in response to SS.

Cyclic strain induces dermal fibroblasts orientation through integrin ?_1-focal adhesion kinase pathway / 第三军医大学学报

Objective To assess the effects of cyclic stretch on fibroblast orientation in order to find the appropriate cyclic stretch to cause maximum fibroblast orientation and to explore the mechanism of cell signalling since cells are known to orient in response to the application of mechanical forces. Methods Human forehead dermal fibroblasts were seeded onto collagen coated flexible membranes. Membranes were then deformed at 10 cycles per minute by the application of 135 mmHg subatmospheric pressure. This corresponded to strain levels of 0% to 24% from the center to extremity of the flexible membrane. Cells orientation angles were studied by inverted microscopy. Integrin ?1 distribution were studied with immunocytochemical staining and confocal microscopy. Integrin ?1 expression and focal adhesion kinase ( FAK) phosphorylation were analyzed with Western blot analysis. Results A minimum of 15% cell stretch was required to significantly stimulate the fibroblast orientation response. Cyclic stretch induced integrin ?1 redistribution and FAK phosphorylation. Incubation of cells with anti-integrin ?1 prior to the application of stretch abrogated fibroblast orientation and FAK phosphorylation. Conclusion Fibroblast orientation in response to cyclic stretch is mediated at least in part by integrin ?1 through phosphorylation of FAK.