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.

Cyclic Stretch Promotes Migration of MC3T3-E1 Cells via Increasing Runx2 Expression / 医用生物力学

Objective To investigate the effects of cyclic stretch on migration of MC3T3-E1 cells and its related mechanism. Methods The strain loading system was used to stretch MC3T3-E1 cells cultured in vitro with 15% amplitude, to simulate the mechanical condition in vivo. The wound healing assay was used to detect the migration of MC3T3-E1 cells. Western blotting was used to test Runx2 expression. RNA interfering was used to decrease Runx2 expression. Results Cyclic mechanical stretch with 15% amplitude, 1.25 Hz frequency and lasting for 24 hours could promote the migration of MC3T3-E1 cells and increase the expression level of Runx2. Runx2 interference inhibited the migration of MC3T3-E1 cells in static culture condition. Interference with Runx2 expression in MC3T3-E1 cells could partially reduce the positive effect of cyclic mechanical stretch on cell migration. Conclusions Cyclic stretch can promote the migration of MC3T3-E1 cells, and Runx2 may play an important role in this process. This study provides experimental basis for finding innovative clinical treatment method to promote fracture healing.

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.

Characteristics of vascular remodeling in pregnant hypertensive rats induced by L-NAME / 医用生物力学

Objective To elucidate the characteristics of vascular remodeling in pregnant hypertensive rats.Methods Pregnant rats were induced by L-nitro-arginine methylester (L-NAME) to build hypertension models and normal pregnant rats were used as control.Using a programmable sphygmomanometer,the blood pressure was recorded with the tail-cuff method to ensure the hypertension model was successfully replicated.The changes of mean shear stress were determined after the blood viscosity,the average blood flow and the inner diameter in left common carotid artery were measured.To analyze the degree of arterial remodeling,the protein expression levels of collagen Ⅰ (Col Ⅰ) and Ⅲ (Col Ⅲ) were detected by Western blotting,and the media thickness,the inner diameter,the opening angel both in thoracic aorta and carotid artery were determined.Results The mean shear stress of common carotid artery was reduced by (28.52 ± 3.08) ％ with the blood viscosity increasing and the average blood flow decreasing in pregnant hypertensive rats.Compared with control groups,the ratio of media thickness and inner diameter significantly increased in thoracic aorta and carotid artery,while the opening angel decreased in carotid artery and increased in thoracic aorta.With the expression of Col Ⅰ decreasing and Col Ⅲ increasing,the ratio of Col Ⅰ and Col Ⅲ went an apparent decline.Conclusions The mean shear stress is descending in pregnant hypertensive rat,with the remodeling of thoracic aorta and carotid artery.These results may provide new experimental references for further illustrating pathogenesis of pregnant hypertension.

Mechanics-proteomics progress on vascular mechnobiology / 医用生物力学

The mechanobiological mechanism in vascular homeostasis and vascular remodeling is one of the most important areas in stress-growth research,which is still unclear.Proteomics analysis,which is a high-throughput and systemic technic,is recently combined with biomechanics,bioinformatics and traditional molecular biology,and applied to demonstrate the mechanism of vascular remodeling induced by different kinds of mechanical stresses.These multidisciplinary and integrated technologies give new insights into understanding the mechanobiological mechanism of vascular remodeling and provide novel potential targets of clinical therapy on cardiovascular diseases.During recent years,the Institute of Mechanobiology & Medical Engineering of Shanghai Jiao Tong University has launched systematic researches with 3 steps:phenomenon exploration with mechanobiological experiments,bioinformatics analysis,and biological and experimental verifications,which established a potential mechanotransduction networks and more than 60 kinds of the novel mechanoresponsive molecules as well.Further researches were performed to demonstrate the role of these molecules in regulation of cellular functions under different kinds of mechanical stimuli.This paper reviews the recent progresses in vascular proteomics and the relative researches on mechanobiology.Researches based on mechanics-proteomics technics may contribute to the understanding of the pathogenesis of cardiovascular diseases,and provide novel therapeutic targets for vascular remodeling during hypertension and atherosclerosis.

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.

Expression of RhoGDIα in aortae of hypertensive rats / 解剖学报

Objective To evaluate the role of angiotensin Ⅱ(AngⅡ) signal passway on the expression of Rho GDP dissociation inhibitor alpha (RhoGDIα) in hypertensive rats. Methods Protein and mRNA expressions of RhoGDIα in aortae of 4, 12 and 18 week-old spontaneously hypertensive rats (SHR, n = 4) and Wistar Kyoto rats (WKY, n= 4) were examined by Western blotting and real-time PCR. Aortas from SHR and WKY were analyzed using immonuchemical staining to locate the RhoGDIα in the aorta. The RhoGDIα expression in aorta of hypertensive rat model of aorta coarctation (ACR, n = 6) was also analyzed using Western blotting. Furthermore, The effect of mechanical strain at 10 % elongation on expression of RhoGDIα in vascular smoothmuscle cells (VSMCs) in the presence or absence of L-158809, an antagonist for AngⅡ type 1 receptor, was also evaluated by Western blotting. Results No significant difference of RhoGDIα expression was found between SHR and WKY at 4-week-old and 12-week-old. However, in 18-week-old group, RhoGDIα was significantly highly expressed in SHR than that of WKY at both mRNA and protein levels. RhoGDIα was located in the media of the aorta. Expression of RhoGDIα protein was upregulated in aortas of ACR at 2 and 4 weeks as compared with the controls. The expression of RhoGDIα in VSMCs was inhibited by mechanicalstrain at 10 % elongation, and further decreased by treatment of L-158809. Conclusion RhoGDIα is upregulated in aortae of the hypertensive rats. AngⅡ signal passway may be involved in the process of regulating expression of RhoGDIα.

The effect of imidazoline receptor agonist-moxonidine on the His bundle electrogram in vivo / 西安交通大学学报(医学版)

Objective To study the effect of moxonidine (Mox) on the His bundle electrogram (HBE) of normal rabbits. Methods A total of 24 healthy rabbits were randomly divided into four groups: control group, small dose of Mox (0.1 mg?kg -1), medium dose of Mox (0.3 mg?kg -1) and large dose of Mox (0.9 mg?kg -1). The electrode catheter was inserted from the right carotid artery to record the HBE. The HBE and the synchronism surface ECG were recorded before and after intravenous injection. Results In normal rabbits, the R-R interphase, P-R interphase of the ECG and the H-V interphase of the HBE were prolonged in a dose-dependent manner after intravenous injection of Mox. Mox exerted no significant influence on the A-H interphase. Conclusion ① Mox decreases the heart rate of rabbits in a dose-dependent manner in vivo. ② Mox dose-dependently prolongs the P-R interphase of the surface ECG and the H-V interphase of HBE. This indicates that Mox mainly acts on the intraventricular conducting system.