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.

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 in left common carotid artery were determined after the blood viscosity, the average blood flow and the inner diameter 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 were determined in both thoracic aorta and carotid artery. Results The mean shear stress of common carotid artery in pregnant hypertensive rats 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 was significantly increased in thoracic aorta and carotid artery, while the opening angel decreased in carotid artery while 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, and the remodeling of thoracic aorta and carotid artery are found in pregnant hypertensive rats. These results may provide new experimental references for further illustrating the pathogenesis of pregnant hypertension.

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.

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.

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.

The role of microRNA-133b in proliferation of vascular smooth muscle cells induced by endothelial cells under low shear stress / 医用生物力学

Objective To investigate the role of microRNAs (miRs) in the proliferation of vascular smooth muscle cells （VSMCs）induced by endothelial insulin-like growth factor-1 (IGF-1) under low shear stress (LowSS). Methods Endothelial cells (ECs) and VSMCs were co-cultured and exposed to normal shear stress (NSS, 1.5 Pa) and LowSS (0.5 Pa) for 12 h with parallel plate flow chamber system, respectively. Real-time PCR was used to examine the expression levels of miRs. The target genes of miR-133b were predicted by multiple algorithms. The expression of polypyrimidine tract binding protein 1 (Ptbp1) and N-myc downstream regulated 1 (Ndrg1) in VSMCs was detected by Western blotting. The VSMC proliferation was detected by EdU flow cytometry assay. Results After treated with recombinant IGF-1, the expression of both miR-133b and miR-378a in VSMCs was increased. Compared with NSS, LowSS significantly induced the expression of miR-133b in the co-cultured VSMCs, but had no obvious effect on miR-378a. In VSMCs, the protein and mRNA levels of Ptbp1 and Ndrg1 were down-regulated by miR-133b mimics. miR-133b inhibitor up-regulated the mRNA levels of Ptbp1 and Ndrg1. miR-133b overexpression promoted the proliferation of VSMCs significantly. Conclusions IGF-1 secreted by ECs in response to LowSS can upregulate the expression of miR-133b in the co-cultured VSMCs, which subsequently depresses the expression of Ptbp1 and Ndrg1, and induces the proliferation of VSMCs eventually. The research findings provide a potential new target for cardiovascular disease therapy.

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.

The role of microRNA-34a in low shear stress- induced proliferation of vascular smooth muscle cells / 医用生物力学

Objective To investigate the role of microRNA-34a (miR-34a) in the proliferation of vascular smooth muscle cells (VSMCs) induced by low shear stress (LowSS). Methods Using co-culture parallel plate flow chamber system, endothelial cells (ECs) and VSMCs were co-cultured and applied with normal shear stress (1.5 Pa) and LowSS (0.5 Pa) for 12 h. The expression of proliferating cell nuclear antigen (PCNA) in the co-cultured VSMCs was detected by Western blotting to determine the proliferation capacity of VSMCs. Real-time PCR was used to examine the miR levels of miR-34a in the co-cultured VSMCs. The target proteins of miR-34a were predicted by TargetScan, miRWalk and some other websites. Western blotting was used to detect expression of Forkhead box j2 (Foxj2) in the co-cultured VSMCs. Mimics and inhibitor were used to up-regulate or inhibit the expression of miR-34a, and then the expression of Foxj2 and PCNA was detected by Western blotting to verify the regulation relationship between miR 34a and Foxj2. Results Compared with NSS, LowSS promoted the PCNA expression and significantly up-regulated the miR-34a expression in the co-cultured VSMCs. Foxj2 was predicted to be the downstream target protein of miR-34a by TargetScan, miRWalk and some other websites. Foxj2 expression decreased significantly in the co-cultured VSMCs under LowSS application. Under static condition, the expression of Foxj2 obviously decreased and the expression of PCNA obviously increased by up-regulating miR-34a expression in VSMCs. While inhibiting the expression of miR-34a in VSMCs would result in a significant increase in the expression of Foxj2 and a significant decrease in the expression of PCNA. Conclusions LowSS can promote the proliferation of VSMCs by regulating miR-34a and target protein Foxj2 in the co-cultured VSMCs. This research finding will provide new mechanobiological experimental reference for further illustrating the pathogenesis of atherosclerosis and finding the therapeutic targets for drugs.

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.

The role of Rab28 and ERK in low shear stress induced migration of vascular smooth muscle cells / 医用生物力学

Objective To study the role of abnormally changed migration of vascular smooth muscle cells (VSMCs) induced by low shear stress (LowSS) in vascular remodeling during atherosclerosis as well as the molecular mechanism involved in this process. Methods By using comparative proteomic analysis with two-dimensional electrophoresis combined with mass spectrometry, differential protein expression profiles of cultured vascular tissues under normal shear stress (NSS) (1.5 Pa) and LowSS (0.5 Pa) were studied. Using endothelial cells (ECs) and VSMCs co-cultured parallel plate flow chamber system, two levels of shear stress i.e. LowSS and NSS, were applied, respectively. Western blot was used to detect the protein expressions of Rab28 and phosphor-ERK. Transwell system was used to detect the migration ability of VSMCs. After using RNA interference and ERK inhibitor PD98059 to decrease the expressions of Rab28 and phosphor-ERK, respectively, the migration ability of VSMCs was observed again. Results The expression of Rab28 in the cultured rat aorta was significantly up-regulated by the LowSS (0.5 Pa) application in comparison with the NSS (1.5 Pa). The migration, expressions of Rab28, and phosphorylation of ERK in VSMCs were significantly increased by the LowSS application. Target RNA interference of Rab28 significantly decreased the migration of VSMCs, but had no specific effect on the phosphorylation of ERK. Target inhibitor of ERK, PD98059, significantly decreased both the migration and Rab28 expression in VSMCs. Conclusions The LowSS may increase the phosphorylation of ERK and then increase the expression of Rab28 in VSMCs, which subsequently modulate VSMC migration during vascular remodeling. The investigation on the role of Rab28 and its signal path in LowSS-regulated VSMCs as well as the molecular mechanism might provide a biomechanical reference for understanding the pathogenesis of vascular remodeling during atherosclerosis and finding the therapeutic target of new drugs.

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.

Shear stress modulates the proliferation of vascular smooth muscle cells via receptor for activated C kinase 1 / 医用生物力学

Objective To investigate the role of receptor for activated C kinase 1 (RACK1) in vascular smooth muscle cells (VSMCs) proliferation modulated by co-cultured endothelial cells (ECs) and shear stress. Methods Using EC/VSMC co-cultured parallel plate flow chamber system, two levels of shear stress, i.e. low shear stress (LowSS, 0.5 Pa) and normal shear stress (NSS, 1.5 Pa), were applied for 12 h. BrdU ELISA was used to detect the proliferation of VSMCs, and Western blot was used to detect the protein expressions of RACK1 and phosphor-Akt. Under the static condition, RNA interference was used to suppress the expression of RACK1 in VSMCs, and then the proliferation of VSMCs and expressions of RACK1 and phosphor-Akt were detected. By using co-culture model (ECs/VSMCs) and separated culture model (ECs//VSMCs), the effect of ECs on expressions of RACK1 and phosphor-Akt in VSMCs was further analyzed. Results Comparative proteomic analysis revealed that LowSS increased the expression of RACK1 in rat aorta. In vitro experiments showed that LowSS induced the proliferation, expressions of RACK1 and phospho Akt in VSMCs co-cultured with ECs. Target RNA interference of RACK1 significantly decreased the proliferation of VSMCs, and the phosphorylation of Akt. In comparison with ECs//VSMCs (separated culture) group, the expression of RACK1 and phosphor-Akt were both up-regulated in the VSMCs co-cultured with ECs (ECs/VSMCs group). Conclusions The expression of RACK1 in VSMCs was modulated by shear stress and neighboring ECs, which might induce cellular proliferation via PI3K/Akt pathway. The investigation on VSMC proliferation and the involved biomechanical mechanism will contribute to understanding and help preventing the pathogenesis and progress of atherosclerosis.

Effect from different pore sizes of co-culture inserts on the permeability of platelet derived growth factor / 医用生物力学

Objective To investigate the effect from different pore sizes of co culture inserts on the permeability of biomacromolecules through polyethylene terephthalate (PET) membrane so as to solve the key technology problem in mechanobiology experiment on vascular cells. Methods Inserts with 0.4 μm and 1.0 μm pores on the PET membrane were studied using flow chamber system. Low shear stress was subjected to the co-cultured system of endothelial cell (EC)/vascular smooth muscle cell (VSMC) and the concentration of platelet-derived growth factor BB (PDGF-BB) was detected by ELISA. Under the static condition, vascular cells were cultured on the plate (with no cell on PET membrane), on the outer side of PET membrane, and on the both sides of PET membrane, respectively. Then the recombinants PDGF-BB (rPDGF-BB) were added on the different sides of PET membrane. Western blotting was used to detect the change in expressions of p-ERK1/2, p-Akt and Lamin after cells were stimulated by rPGDF BB. Results After low shear stress subjection for 12 h, the concentration of PDGF-BB in the medium from VSMC side was significantly higher than that from EC-side. rPDGF-BB passed through 0.4 μm and 1.0 μm pores on the PET membrane and modulated expressions of p-ERK1/2, p-Akt and Lamin A in cells cultured on the opposite side of PET membrane and cells cultured on the plate separately. When cells were cultured on the both sides of PET membrane, rPDGF-BB only stimulated cells cultured on the same side of 0.4 μm pores on PET membrane, but had no specific effect on cells cultured on the opposite side. Conclusions PET membrane with both 0.4 μm and 1.0 μm pores was permeable to PDGF-BB, and cells cultured on the membrane could affect the permeability. The efficiency of PDGF BB passing through 0.4 μm pores was significantly repressed with cells cultured on the both sides, which was more similar to that in vivo.

Cyclic strain promotes migration of human periodontal ligament cell via extracellular signal-regulated kinase (ERK) signaling pathway / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To study the effect of cyclic strain on migration of human periodontal ligament cell(hPDLC) and underlying mechanism.</p><p><b>METHODS</b>The cultured hPDLC were subjected to 10% or 20%-elongation magnitude cyclic strain at frequency of 0.1 Hz by FX-4000T system for 6 or 24 hours-duration respectively, while the static group serves as control. hPDLC migration was assayed by wound healing method. The expressions of matrix metalloproteinases-9 (MMP-9) and p-ERK1/2 in hPDLC without or with cyclic strain were analyzed by Western blotting. To investigate the effect of ERK signaling pathway and MMP-9 on migration of hPDLC, the cells were incubated with PD98059, a specific extracellular signal-regulated kinase (ERK) kinase inhibitor, or doxycycline, a MMP inhibitor. Then the expressions of p-ERK1/2 and MMP-9 and hPDLC migration were analyzed.</p><p><b>RESULTS</b>In wound healing tests, the migration of hPDLC exposed to 10% or 20%-cyclic strain at 0.1 Hz-frequency for 6 hours was not apparent but became significantly different for 24 hours (P < 0.05) compared to control. Furthermore, the 20%-elongation magnitude of cyclic strain had more remarkable effect on migration of hPDLC than 10%-elongation magnitude at 24 hours-duration (P < 0.05). Cyclic strain obviously increased the expression of MMP-9 in hPDLC (P < 0.05). PD98059 could repress not only the activation of p-ERK1/2 but also the expression of MMP-9 induced by cyclic strain in hPDLC. The migration of hPDLC enhanced by cyclic strain was repressed by DOX or PD98059 in wound healing tests.</p><p><b>CONCLUSIONS</b>Cyclic strain promotes the migration of hPDLC through activating ERK signaling pathway and inducing the expression of MMP-9.</p>

Shear stress and vascular smooth muscle cells modulate the proliferation of endothelial cells via TGFβ1 and p-Akt pathways / 医用生物力学

Objective To investigate the effects of shear stress and vascular smooth muscle cells (VSMCs) on the proliferation of endothelial cells (ECs) and the molecular mechanism involved in. Method Using parallel-plate flow chamber system, normal shear stress of 15 dyn/cm2(1 dyn=10-5 N) was applied to ECs cultured singly and co cultured with VSMCs respectively. Then, the expression of PCNA, a molecule representing cell proliferation ability, and phosphorylation of Akt were analyzed by Western blotting in order to investigate the roles of shear stress and VSMCs in EC proliferation. Under the static condition, the expressions of PCNA and p-Akt were analyzed in ECs co-cultured with VSMCs with and without physical contact. And then TGFβ1 neutralizing antibody was employed to demonstrate the contribution of TGFβ1 in VSMCs induced EC proliferation. Results Normal shear stress decreased EC proliferation and Akt phosphorylation. VSMCs increased EC proliferation and Akt phosphorylation in both co-culture conditions with and without physical contact. Normal shear stress partly reversed the increase of proliferation and Akt phosphorylation in ECs with physical contact to VSMCs, and TGFβ1 neutralizing antibody exerted the similar effects in ECs without physical contact to VSMCs. Conclusions Normal shear stress is a protective factor with its inhibitory effect on EC proliferation. VSMCs induced EC proliferation via TGFβ1 and p Akt pathways by paracrine model.