Cyclic Mechanical Stretch Research Articles

182 EXPRESSION OF THE PROANGIOGENIC FACTOR CYSTEINE-RICH PROTEIN 61 GENE IN MECHANICALLY STRAINED BLADDER SMOOTH MUSCLE CELLS REQUIRES THE COMBINED ACTIVITIES OF THE MYOCARDIN-RELATED TRANSCRIPTION FACTOR AND HISTONE ACETYL TRANSFERASE

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology I1 Apr 2010182 EXPRESSION OF THE PROANGIOGENIC FACTOR CYSTEINE-RICH PROTEIN 61 GENE IN MECHANICALLY STRAINED BLADDER SMOOTH MUSCLE CELLS REQUIRES THE COMBINED ACTIVITIES OF THE MYOCARDIN-RELATED TRANSCRIPTION FACTOR AND HISTONE ACETYL TRANSFERASE Mary Hanna, Haibo Liu, and Brahim Chaqour Mary HannaMary Hanna More articles by this author , Haibo LiuHaibo Liu More articles by this author , and Brahim ChaqourBrahim Chaqour More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.238AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The cellular components of the urinary bladder elicit adaptive responses to pressure overload acutely by remodeling their cytoskeletal structures and chronically through hypertrophic growth of smooth muscle cells (SMCs). The long term responses are associated with the activation of angiogenesis to improve tissue perfusion which, otherwise, becomes impaired as a result of a mismatch between the smooth muscle mass and capillary density, leading to hypoxia/ischemia, altered muscle contractility and organ failure. Neoangiogenesis is promoted by mechano-responsive angiogenic factors such as cysteine-rich protein 61 (Cyr61) which enhances sprouting of new blood vessels and protects against oxidative stress. In this study, we examined the expression and regulation of the Cyr61 gene by mechanical forces in bladder SMCs. METHODS Cultured human bladder SMCs were subjected to cyclic mechanical stretch using a mechanical device designed to impart a biaxial strain to the cells. Partial urethral obstruction of the bladder was created in female Sprague Dawley rats by loosely tying the urethra. The expression and activation of the Cyr61 gene was examined by real-time PCR, transfection and chromatin immunoprecipitation (ChIP) assays. RESULTS Mechanical strain-dependent induction of the Cyr61 gene involves signaling cascades through RhoA-mediated actin remodeling and the p38 stress-activated protein kinase (SAPK). Actin signaling controls serum response factor (SRF) activity via SRF interaction with the myocardin-related transcriptional activator (MRTF)-A and its tethering to a single CArG-box sequence within the Cyr61 promoter. Such activity was abolished in mechanically stimulated mouse MRTF-A null cells or upon inhibition of cyclic AMP responsive element (CREB)-binding protein (CBP) histone acetyl transferase (HAT). Mechanical strain induced CBP-mediated acetylation of histones 3 and 4 at the SRF binding site. Inhibition of p38 SAPK reduced CBP HAT activity and its recruitment to the SRF-MRTF-A complex. Similarly, mechanical overload-induced Cyr61 gene expression in obstructed bladders was associated with nuclear localization of MRTF-A and enrichment of the Cyr61 promoter with both MRTF-A and acetylated histone H3. CONCLUSIONS Signal-controlled activation of SRF, MRTF-A and CBP provides a novel connection between mechanical forces and angiogenic gene expression in bladder smooth muscle. Brooklyn, NY© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e72-e73 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Mary Hanna More articles by this author Haibo Liu More articles by this author Brahim Chaqour More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Angiotensin II signalling regulates skeletal muscle growth and myoblast chemotaxis

The regulatory role of angiotensin II (Ang II) in mediating skeletal muscle hypertrophy is largely unknown. We examined the impact of Ang II on myoblast chemotaxis using transwell assays. Exogenous treatment of Ang II significantly increased the migratory capacity of both C2C12 and primary myoblasts by 30 and 43% respectively, while Ang II receptor knockout myoblasts (AT1a−/−) demonstrated a significant attenuation of basal migration (38% of control) and did not respond to exogenous Ang II treatment. Ang II appeared to increase migration by inducing MMP2 activity as gelatin zymography analysis of cell culture media and lysates revealed a ~40% increase in gelatinase activity and pharmacological inhibition of MMP2 abolished the Ang II mediated increase in myoblast migration. Ang II also appeared to signal in a paracrine fashion to induce migration as 4h of cyclic mechanical stretch of myoblasts enhanced migration of co‐cultured myoblasts. To test the involvement of Ang II signaling during skeletal muscle regeneration in vivo, control and At1a−/− mice were subjected to cardiotoxin injury. In comparison to controls, at 14 and 21 days following injury, At1a−/− mice demonstrated 34 and 25% reductions in cross sectional area respectively. Taken together, these results demonstrate that Ang II modulates skeletal muscle fibre growth by regulating myoblast chemotaxis through MMP2.

ATF3 is activated in ventilator induced lung injury

IntroductionMechanical ventilation (MV) has been shown to cause ventilator‐induced lung injury (VILI). We wanted to identify novel molecular targets involved in reducing the harmful effects of MV (VILI).MethodsTo identify potential novel genes and proteins involved in VILI we i) performed microarray analysis on previously published VILI animal model data, ii) exposed human bronchial epithelial cells (Beas‐2b) in vitro to mechanical cyclic stretch (30 cycles/min for 4 hours), iii) performed western blot analysis on these stretched and non‐stretched cells, and iv) validated the regulation of the newly discovered gene(s) in vivo using wild type (WT) and ATF3 (Activator transcription factor 3) knock‐out mice (ATF3 KO). These two groups of mice were exposed to either injurious or non‐injurious ventilation strategies in combination with LPS or saline.ResultsATF3 was identified by microarray analysis as one of the main genes involved in VILI. The presence of this factor was validated in vitro in the mechanically cyclic‐stretched Beas‐2b cells. Western blot assays confirmed expression of ATF3 in the stretched cells and their absence in the static cells. We further confirmed the regulation of ATF3 using WT and ATF3 KO mice in a model of VILI in vivo. ATF3 KO mice exposed to LPS in combination with injurious MV had more inflammation than WT mice of similar treatment or mice treated with saline.ConclusionExpression of ATF3 is stretch dependent and this gene seems to confer protection in combination with MV. Specific targeting of this factor in clinical settings could reduce VILI in patients receiving MV.

Cyclic stretch enhances the expression of Toll-like Receptor 4 gene in cultured cardiomyocytes via p38 MAP kinase and NF-κB pathway

BackgroundToll-like receptor 4 (TLR4) plays an important role in innate immunity. The role of TLR4 in stretched cardiomyocytes is not known. We sought to investigate whether mechanical stretch could regulate TLR4 expression, as well as the possible molecular mechanisms and signal pathways mediating the expression of TLR4 by cyclic mechanical stretch in cardiomyocytes.MethodsNeonatal Wistar rat cardiomyocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation at 60 cycles/min. Western blot, real-time polymerase chain reaction, and promoter activity assay were performed. In vitro monocyte adhesion to stretched myocyte was detected.ResultsCyclic stretch significantly increased TLR4 protein and mRNA expression after 2 h to 24 h of stretch. Addition of SB203580, TNF-α antibody, and p38α MAP kinase siRNA 30 min before stretch inhibited the induction of TLR4 protein. Cyclic stretch increased, while SB203580 abolished the phosphorylated p38 protein. Gel shifting assay showed significant increase of DNA-protein binding activity of NF-κB after stretch and SB203580 abolished the DNA-protein binding activity induced by cyclic stretch. DNA-binding complexes induced by cyclic stretch could be supershifted by p65 monoclonal antibody. Cyclic stretch increased TLR4 promoter activity while SB203580 and NF-κB siRNA decreased TLR4 promoter activity. Cyclic stretch increased adhesion of monocyte to cardiomyocytes while SB203580, TNF-α antibody, and TLR4 siRNA attenuated the adherence of monocyte. TNF-α and Ang II significantly increased TLR4 protein expression. Addition of losartan, TNF-α antibody, or p38α siRNA 30 min before Ang II and TNF-α stimulation significantly blocked the increase of TLR4 protein by AngII and TNF-α.ConclusionsCyclic mechanical stretch enhances TLR4 expression in cultured rat neonatal cardiomyocytes. The stretch-induced TLR4 is mediated through activation of p38 MAP kinase and NF-κB pathways. TLR4 up-regulation by cyclic stretch increases monocyte adherence.

Regulation of resistin by cyclic mechanical stretch in cultured rat vascular smooth muscle cells

Resistin has a potential role in atherosclerosis; however, the molecular mechanism underlying the increase in resistin expression in atherosclerosis remains unclear. As mechanical stretch plays an important role in atherosclerosis, in the present study we sought to investigate the cellular and molecular mechanisms underlying the regulation of resistin by cyclic mechanical stretch in VSMCs (vascular smooth muscle cells). VSMCs from thoracic aorta of adult Wistar rats were cultured and subjected to cyclic stretch. Cyclic mechanical stretch significantly increased resistin protein and mRNA expression as compared with control cells without stretch. The specific p38 MAPK (mitogen-activated protein kinase) inhibitor SB203580, the antioxidant N-acetylcysteine and p38 MAPK siRNA (small interfering RNA) attenuated the induction of resistin protein by cyclic stretch. Cyclic stretch significantly increased the phosphorylation of p38 MAPK, whereas pre-treatment with SB203580 and N-acetylcysteine significantly inhibited this effect. Cyclic stretch significantly increased ROS (reactive oxygen species) production, and pre-treatment with N-acetylcysteine significantly inhibited stretch-induced ROS production. Cyclic stretch also increased STAT3 (signal transducer and activator of transcription 3)-binding activity and resistin promoter activity, and resistin promoter activity was abolished when STAT3 in the promoter area was mutated. Pre-treatment with SB203580 and N-acetylcysteine significantly attenuated resistin promoter activity induced by cyclic stretch. Cyclic stretch increased the secretion of AngII (angiotensin II) and resistin from cultured VSMCs. Exogenous AngII increased resistin expression, and AngII receptor inhibition attenuated this effect. In conclusion, cyclic mechanical stretch increases resistin expression in cultured rat VSMCs. Stretch-induced resistin expression is mediated through ROS, and the p38 MAPK and STAT3 pathways. Therefore resistin induced by cyclic stretch may contribute to the pathogenesis of atherosclerosis under haemodynamic overload.Key

Cyclical Mechanical Stretch Enhances the Proinflammatory but not Profibrotic Responses of Asthmatic Fibroblasts

RATIONALE: Airway remodeling in asthma is characterized by increased smooth muscle mass and deposition of extracellular matrix proteins (ECMs). Tissue damage and repair drives the remodeling in asthma. We postulated that mechanical strain during cough or wheezing may activate submucosal fibroblasts to contribute to remodeling responses.

Cyclic Mechanical Stretch Decreases Cell Migration by Inhibiting Phosphatidylinositol 3-Kinase- and Focal Adhesion Kinase-mediated JNK1 Activation

Epithelial cell migration during wound healing requires coordinated signaling pathways that direct polarization of the leading and trailing ends of the cells, cytoskeletal organization, and remodeling of focal adhesions. These inherently mechanical processes are disrupted by cyclic stretch (CS), but the specific signaling molecules involved in this disruption are not well understood. In this study, we demonstrate that inhibition of phosphatidylinositol 3-kinase (PI3K) or expression of a dominant-negative form of PI3K caused inhibition of airway epithelial cell wound closure. CS caused a sustained decrease in activation of PI3K and inhibited wound healing. Expression of constitutively active PI3K stimulated translocation of Tiam1 to the membrane, increased Rac1 activity, and increased wound healing of airway epithelial cells. Increased Rac1 activity resulted in increased phosphorylation of JNK1. PI3K activation was not regulated by association with focal adhesion kinase. Restoration of efficient cell migration during CS required coexpression of constitutively active PI3K, focal adhesion kinase, and JIP3.

High concentrations of vascular endothelial growth factor reduce stretch‐induced apoptosis of alveolar type II cells

Vascular endothelial growth factor (VEGF) has protective as well as injurious effects in ARDS/acute lung injury. The influence of VEGF was investigated in a model of stretch-induced apoptosis. High-amplitude mechanical stretch induced the secretion of VEGF. High VEGF concentrations may prevent stretch-induced apoptosis by restoring stretch-impaired phospatidylinositol-3 kinase signalling. Vascular endothelial growth factor (VEGF) is strongly expressed in the alveolar epithelium. VEGF has been shown to exhibit protective as well as injurious effects in ARDS/acute lung injury. We therefore investigated the influence of VEGF in a model of stretch-induced apoptosis. Isolated rat alveolar type II (ATII) cells were subjected to high-amplitude cyclic mechanical stretch (40 per minute, 30% change in surface area) for 24 h. VEGF gene expression was investigated by real-time reverse transcription-PCR. Concentrations of VEGF in culture supernatants of stretched cells were determined by ELISA. Apoptosis of cells following stretching was assessed by flow cytometry. Vascular endothelial growth factor gene expression increased during the first 4 h of stretching and then declined to a similar level to that of static control cells. VEGF concentrations in cell supernatants increased in response to mechanical stretch, as compared with those in supernatants of static control cells. Incubation of ATII cells with higher concentrations of VEGF (50 ng/mL) during stretching inhibited apoptosis, presumably by restoring stretch-impaired phosphatidylinositol-3 kinase signalling. However, blocking free VEGF in the supernatant with an anti-VEGF antibody did not influence stretch-induced apoptosis. These findings suggest that high-amplitude mechanical stretch induced secretion of VEGF, which in high concentrations, may prevent stretch-induced apoptosis. In this model, however, the protective influence of VEGF was not essential for survival of ATII cells subjected to high-amplitude mechanical stretch.

Nephron-deficient Fvb mice develop rapidly progressive renal failure and heavy albuminuria involving excess glomerular GLUT1 and VEGF

Reduced nephron numbers may predispose to renal failure. We hypothesized that glucose transporters (GLUTs) may contribute to progression of the renal disease, as GLUTs have been implicated in diabetic glomerulosclerosis and hypertensive renal disease with mesangial cell (MC) stretch. The Os (oligosyndactyly) allele that typically reduces nephron number by ∼50%, was repeatedly backcrossed from ROP (Ra/+ (ragged), Os/+ (oligosyndactyly), and Pt/+ (pintail)) Os/+ mice more than six times into the Fvb mouse background to obtain Os/+ and +/+ mice with the Fvb background for study. Glomerular function, GLUT1, signaling, albumin excretion, and structural and ultrastructural changes were assessed. The FvbROP Os/+ mice (Fvb background) exhibited increased glomerular GLUT1, glucose uptake, VEGF, glomerular hypertrophy, hyperfiltration, extensive podocyte foot process effacement, marked albuminuria, severe extracellular matrix (ECM) protein deposition, and rapidly progressive renal failure leading to their early demise. Glomerular GLUT1 was increased 2.7-fold in the FvbROP Os/+ mice vs controls at 4 weeks of age, and glucose uptake was increased 2.7-fold. These changes were associated with the activation of glomerular PKCβ1 and NF-κB p50 which contribute to ECM accumulation. The cyclic mechanical stretch of MCs in vitro, used as a model for increased MC stretch in vivo, reproduced increased GLUT1 at 48 h, a stimulus for increased VEGF expression which followed at 72 h. VEGF was also shown to act in a positive feedback manner on MC GLUT1, increasing GLUT1 expression, glucose uptake and fibronectin (FN) accumulation in vitro, whereas antisense suppression of GLUT1 largely blocked FN upregulation by VEGF. The FvbROP Os/+ mice exhibited an early increase in glomerular GLUT1 leading to increased glomerular glucose uptake PKCβ1, and NF-κB activation, with excess ECM accumulation. A GLUT1–VEGF–GLUT1 positive feedback loop may play a key role in contributing to renal disease in this model of nondiabetic glomerulosclerosis.

Effect of mitogen-activated protein kinase kinase 6-p38 alpha signal pathway on receptor for advanced glycation end-product expression in alveolar epithelial cells induced by mechanical stretch

To investigate the role of mitogen-activated protein kinase kinase 6 (MKK6) p38 alpha in mechanical stretch induced receptor for advanced glycation end-product (RAGE) expression in alveolar epithelial cells (A549). Recombinant plasmids were transfected into A549 cells with liposome DOTAP. A549 cells transfected with p38 alpha (AF)/pGFP and MKK6b (E)/pGFP plasmids were taken as treated groups, while the groups that transfected with pcDNA3 plasmid and pGFP plasmid served as blank transfection group and control group, respectively. All the groups were then cultured in 6-well Bioflex cell culture plates and exposed to cyclic mechanical stretch at 20% elongation. The infection and expression of gene were assessed by Western blotting analysis. The activity of p38 kinase in A549 cells and the expression of RAGE protein and mRNA were observed. The transfection of MKK6b (E) led to a markedly increase in p38 kinase activity compared with control group. In contrast, the transfection of p38 alpha (AF) significantly inhibited p38 kinase activity. Compared with control group, stretch markedly increased RAGE protein and mRNA expression in MKK6b (E) transfected cells, while it markedly decreased RAGE expression in p38 alpha (AF) transfected cells. MKK6 p38 alpha signaling pathway regulates the expression of RAGE induced by mechanical stretch in A549 cells.

Mechanical stretch-induced RhoA activation is mediated by the RhoGEF Vav2 in mesangial cells

Increased intraglomerular pressure is an important hemodynamic determinant of glomerulosclerosis, and can be modelled in vitro by exposing mesangial cells (MC) to cyclic mechanical stretch. We have previously shown that the GTPase RhoA mediates stretch-induced fibronectin production. Here we investigate the role of the RhoGEF Vav2 in the activation of RhoA by stretch. Primary rat MC were exposed to 1Hz cyclic stretch, previously shown to induce maximal RhoA activation at 1min. Total Vav2 tyrosine phosphorylation and specific phosphorylation on Y172, required for activation, were increased by 1min of stretch. Overexpression of dominant-negative Vav2 Y172/159F in COS-1 cells or downregulation of Vav2 by siRNA in MC prevented stretch-induced RhoA activation. Vav2 is known to be activated in response to growth factors, and we have previously shown the epidermal growth factor receptor (EGFR) to be transactivated by stretch in MC. Both Vav2 Y172 phosphorylation and RhoA activation were blocked by the EGFR inhibitor AG1478 and prevented in MC overexpressing kinase inactive EGFR. Stretch led to physical association between the EGFR and Vav2, and this was dependent on EGFR activation. EGFR Y992 phosphorylation, required for growth factor-induced Vav2 phosphorylation, was also induced by stretch. Activation of both Src and PI3K were necessary upstream mediators of stretch-induced Vav2 Y172 phosphorylation and RhoA activation. In summary, stretch-induced RhoA activation is dependent on transactivation of the EGFR and activation of the RhoGEF Vav2. Src and PI3K are both required upstream of Vav2 and RhoA activation.

Expression of Bone‐related Genes in Bone Marrow MSCs after Cyclic Mechanical Strain: Implications for Distraction Osteogenesis

AimUnderstanding the response of mesenchymal stem cells (MSCs) to mechanical strain and their consequent gene expression patterns will broaden our knowledge of the mechanobiology of distraction osteogenesis.MethodologyIn this study, a single period of cyclic mechanical stretch (0.5 Hz, 2,000 μɛ) was performed on rat bone marrow MSCs. Cellular proliferation and alkaline phosphatase (ALP) activity was examined. The mRNA expression of six bone-related genes (Ets-1, bFGF, IGF-II, TGF-β, Cbfa1 and ALP) was detected using real-time quantitative RT-PCR.ResultsThe results showed that mechanical strain can promote MSCs proliferation, increase ALP activity, and up-regulate the expression of these genes. A significant increase in Ets-1 expression was detected immediately after mechanical stimulation, but Cbfa1 expression becameelevated later. The temporal expression pattern of ALP coincided perfectly with Cbfa1.ConclusionThe results of this study suggest that mechanical strain may act as a stimulator to induce differentiation of MSCs into osteoblasts, and that these bone-related genes may play different roles in the response of MSCs to mechanical stimulation.

Characterization of contraction-inducible CXC chemokines and their roles in C2C12myocytes

Physical exercise triggers the release of several cytokines/chemokines from working skeletal muscles, but the underlying mechanism(s) by which skeletal muscles decipher and respond to highly complex contractile stimuli remains largely unknown. In an effort to investigate the regulatory mechanisms of the expressions of two contraction-inducible CXC chemokines, CXCL1/KC and CXCL5/LIX, in contracting skeletal muscle cells, we took advantage of our in vitro exercise model using highly developed contractile C(2)C(12) myotubes, which acquire properties similar to those of in vivo skeletal muscle via manipulation of Ca(2+) transients with electric pulse stimulation (EPS). Production of these CXC chemokines was immediately augmented by EPS-evoked contractile activity in a manner dependent on the activities of JNK and NF-kappaB, but not p38, ERK1/2, or calcineurin. Intriguingly, exposure of myotubes to cyclic mechanical stretch also induced expression of these CXC chemokines; however, a much longer period of stimulation (approximately 12 h) was required, despite rapid JNK phosphorylation. We also demonstrate herein that CXCL1/KC and CXCL5/LIX have the ability to raise intracellular Ca(2+) concentrations via CXCR2-mediated activation of pertussis toxin-sensitive Galpha(i) proteins in C(2)C(12) myoblasts, an action at least partially responsible for their migration and differentiation. Although we revealed a possible negative feedback regulation of their own production in response to the contractile activity in differentiated myotubes, exogenous administration of these CXC chemokines did not acutely influence either insulin-induced Akt phosphorylation or GLUT4 translocation in C(2)C(12) myotubes. Taken together, these data shed light on the fundamental characteristics of contraction-inducible CXC chemokine production and their potential roles in skeletal muscle cells.

Engineered Early Embryonic Cardiac Tissue Increases Cardiomyocyte Proliferation by Cyclic Mechanical Stretch via p38-MAP Kinase Phosphorylation

Cardiomyocyte (CM) transplantation is one therapeutic option for cardiac repair. Studies suggest that fetal CMs display the best cell type for cardiac repair, which can finitely proliferate, integrate with injured host myocardium, and restore cardiac function. We have recently developed an engineered early embryonic cardiac tissue (EEECT) using embryonic cardiac cells and have shown that EEECT contractile properties and cellular proliferative response to cyclic mechanical stretch stimulation mimic developing fetal myocardium. However, it remains unknown whether cyclic mechanical stretch-mediated high cellular proliferation activity within EEECT reflects CM or non-CM population. Studies have shown that p38-mitogen-activated protein kinase (p38MAPK) plays an important role in both cyclic mechanical stretch stimulation and cellular proliferation. Therefore, in the present study, we tested the hypothesis that cyclic mechanical stretch (0.5 Hz, 5% strain for 48 h) specifically increases EEECT CM proliferation mediated by p38MAPK activity. Cyclic mechanical stretch increased CM, but not non-CM, proliferation and increased p38MAPK phosphorylation. Treatment of EEECT with the p38MAPK inhibitor, SB202190, reduced CM proliferation. The negative CM proliferation effects of SB202190 were not reversed by concurrent stretch stimulation. Results suggest that immature CM proliferation within EEECT can be positively regulated by mechanical stretch and negatively regulated by p38MAPK inhibition.

Cyclic stretch‐induced proliferation in rabbit proximal tubular cells involves cPLA2, ERK, and c‐Src

Increased intrarenal pressure, in unilateral uretal obstruction (UUO), is considered a major event leading to persistent tubular cell proliferation. The mechanical signal produced by UUO is assumed to be tubular stretching, but the molecular mechanisms involved in this process are poorly understood. In the present study we investigated whether a "physiological" level of stretch (5%) or a more injurious magnitude of stretch (15%) can induce proliferation in kidney epithelial cells. To produce strain or stretch, primary cultures of rabbit proximal tubular cells were grown in tissue culture wells having collagen‐coated Silastic deformable membrane bottoms and exposed to 5% or 15% stretch for 6 h or 24 h. We found that cyclic stretch significantly induced the proliferation of rabbit proximal tubular cells at both 5% and 15% strain for 24 h. Inhibition of PLA2 by AACOCF3 or with the antisense oligonucleotide treatment designed to silence cPLA2 expression, EK 1/2 by PD98059 and c‐Src by a selective inhibitor for the Src kinase family, PP2 or with a dominant‐negative (DN) mutants of c‐Src, blocked the cyclic stretch‐induced proliferation. Overall, this study demonstrates, for the first time, that cyclic mechanical stretch induces the proliferation of rabbit proximal tubular cells and suppresses their mitogenic affect through the activation of cPLA2, ERK and c‐Src.

Rapamycin reduces high-amplitude, mechanical stretch-induced apoptosis in pulmonary microvascular endothelial cells

Alveolar epithelial and endothelial cell death caused by mechanical over-distension is likely to contribute to ventilator-induced lung injury (VILI). Consequently, the search for cytoprotective interventions is of interest. We investigated the effect of the mTOR inhibitor rapamycin on human pulmonary microvascular endothelial cell (HMVEC-L) viability in a model of high-amplitude mechanical stretch. Cyclic mechanical stretch (30% increase in membrane surface area, cycling frequency 40/min), employed for 24 h induced apoptosis in HMVEC-L. This effect was reduced by rapamycin treatment. Focusing on possible mechanisms of action we demonstrated that the stretch-induced reduction in the anti-apoptotic messenger pAkt could be restored by rapamycin treatment. Furthermore, we observed rapamycin-induced modifications in the HMVEC-L actin cytoskeleton architecture and global cellular f-actin content which functionally resulted in an increased global cellular mechanical stability – as indicated by an increased HMVEC-L osmomechanical resistance – thereby possibly desensitizing HMVEC-L to mechanical stimulation. According to the data from the present study, rapamycin represents a promising cytoprotective agent under mechanically challenging conditions such as mechanical ventilation.

Jamming Dynamics Of Stretch-induced Surfactant Secretion By Alveolar Epithelial Cells

Secretion of molecules by cells is a fundamental process of life that maintains the cellular micro-environment. In the lung, secretion of surfactant by alveolar epithelial type II cells is vital for the reduction of interfacial surface tension, thus preventing lung collapse. We find evidence of complex secretory dynamics of these cells in culture when exposed to cyclic mechanical stretch which is the primary stimulus for surfactant secretion. We find that (a) during and immediately following stretch, cells secreted less surfactant than unstretched cells and (b) cells stretched for 15 minutes secreted significantly more surfactant than unstretched cells after 45 min of rest.

Cyclic stretch induces organization of intercellular communication via polarization of gap junctions in cardiac cells. Effect of angiotensin-II

Aims: Arrhythmia often is found in tissue which exhibits altered localization (lateralization) of intercellular communication channels (gap junctions), thereby altering the electrical network. We examined whether cyclic mechanical stretch (CMS) can affect localization of gap junctions with regard to the cell axis.

Upregulation of cardiac matrix Gla protein expression in response to hypertrophic stimuli

Matrix Gla protein (MGP) expression is increased in cardiac hypertrophy, but the precise mechanisms regulating its expression are unknown. Here we characterized the effect of pressure overload and myocardial infarction in vivo as well as mechanical stretch and hypertrophic agonists in vitro on MGP expression. When angiotensin II (Ang II) was administered by osmotic minipumps, left ventricular (LV) MGP mRNA levels increased significantly from 6 h to 2weeks, whereas intravenous arginine8-vasopressin increased LV MGP mRNA levels within 4 h. During post-infarction remodeling process, MGP mRNA levels were elevated at 24 h (1.3-fold, p<0.05) and the maximal increase was observed at 4 weeks (2.8-fold, p<0.01). Ang II increased MGP mRNA levels 20% (p<0.05) in neonatal rat cardiac myocytes and 40% (p<0.05) in cardiac fibroblasts, whereas endothelin-1 decreased MGP mRNA levels 30% (p<0.01) in myocytes and had no effect in fibroblasts. Cyclic mechanical stretch resulted in reduction of MGP gene expression in both cardiac myocytes and fibroblasts. These results demonstrate that MGP is rapidly upregulated in response to cardiac overload well before the development of LV hypertrophy and post-infarction remodeling process. Our results also suggest that Ang II may be involved in mediating load-induced activation of MGP expression.

Gene Expression Profiles of Neurotrophic Factors in Rat Cultured Spinal Cord Cells Under Cyclic Tensile Stress

An experimental study to investigate the in vitro gene expression of neurotrophic factors and receptors in cultured rat spinal cord cells subjected to cyclic mechanical stretch forces. We evaluated in vitro expression of neurotrophic factors and receptors in cultured rat spinal cord cells under cyclic tensile stress. Application of compressive mechanical stress to the spinal cord results in multiple changes making it difficult to examine the expression of neurotrophic factors and their receptors. There are no in vitro studies that investigated the biologic responses of cultured spinal cord cells to tensile stress. Spinal cord cells were isolated for culture from 15-day Sprague-Dawley rat embryos. We used the FX3000 Flexercell Strain Unit to induce mechanical stress. We analyzed the effects of mechanical stress on cell morphology, mRNA expression levels of various neurotrophic factors, and their immunoreactivities at 0, 2, 6, 12, 24, and 36 hours. Tensile stress for 6 hours resulted in reduction of spinal cord cells and loss of neurites. Cells that survived 24-hours stress showed swollen irregular-shaped soma, bleb formation, and fragmented neurites. The cell survival rate decreased, whereas lactate dehydrogenase release increased significantly at 6 hours. There were significant increases in mRNA expression levels of nerve growth factor, brain-derived neurotrophic factor, trkB, p75 neurotrophin receptor (p75), glial cell line-derived neurotrophic factor, and caspase-9 during the early period after application of tensile stress. Our results suggest survival of spinal cord neuronal cells under injurious tensile stress with increased synthesis and utilization of several neurotrophic factors, receptors, and expression of proteins related to cell apoptosis.