| BackgroundThe vascular wall is permanently subjected to hemodynamic forces in the form of stretch as the result of heart propulsion, and shear stress from circulating blood. Blood flow imposes shear stress on the endothelial cells and mechanical stretch affects all cell types in the vessel wall. Mechanical forces are the main determinant of cardiovascular remodeling. There is considerable literature on the concept that vascular cells respond to mechanical stimuli. In vascular smooth muscle cells (VSMCs), mechanical cyclic stretch can regulate multiple cellular functions including proliferation, migration, and extracellular matrix synthesis. Moderate cyclic stretch, as occurs under physiological conditions, exerts vasoprotective effects and inhibits growth factor-induced proliferation of HASMCs, while increased stretch, as occurs under hypertension, can promote proliferation of HASMCs.Renin-angiotensin system (RAS), especially local vascular RAS activation, plays an important role in the pathogenesis of vascular diseases. Angiotensin-converting enzyme (ACE) and angiotensin II (Ang II) are involved in neointimal thickening, which is mediated by Ang II type1(AT1) receptor signaling events through monocyte chemoattractant protein-1and NAD(P)H oxidase. The role of ACE and Ang II in the remodeling of large and resistance arteries during hypertension are also established.The recently discovered ACE2, the homologue of ACE, has added a new dimension to the traditional RAS, which is constituted by ACE/Ang II/AT1receptor. Accumulating evidence suggests that this new member of the RAS may act as an endogenous negative regulator of RAS. ACE2, in contrast to ACE, degrades Ang II into the Ang-(1-7) and Ang I into the Ang-(1-9), peptides with cardiovascular protective effects, thus it is considered as an endogenous inhibitor of ACE.In vessels, the existence of the new members of the RAS, including ACE2, Ang-(1-7) and Mas, have been established. A recent study found that it is the tissue RAS, in stead of the circulating RAS and inflammatory factors, played a decisive role in hypertensive cardiovascular hypertrophy. ACE2mRNA expression is ubiquitously found in arterial endothelial cells and smooth muscle cells (HASMCs). This relatively restricted expression of ACE2, together with its differential enzyme activity for the key vasoactive peptide Ang II, suggests a distinctive role of ACE2in the local RAS of the vessel.Mechanical stretch can induce AngⅡ release from the endothelium, which is accompanied by elevated ROS levels. On the other hand, as one of the major receptors of AngⅡ, AT1R can be directly activated by mechanical stretch independently of Ang II release. In light of these previous studies, we speculate that mechanical cyclic stretch also modulates the expression of ACE2and its enzyme activity. ACE2may be involved in the effects of stretch on the cellular functions of VSMCs. To test our hypothesis, we performed in vitro and in vivo experiments respectively to examine the direct effect of mechanical stretch on the mRNA and protein expression of ACE2in HASMCs and to identify the role of ACE2in mechanical stretch mediated cellular functions.Objectives1. To investigate whether mechanical stretch can modulate the expression of ACE2in human vascular smooth muscle cells (HASMCs).2. To elucidate the role of ACE2in stretch mediated cellular functions of HASMCs.3. To verify the influence of mechanical forces on the expression of ACE2in vivo.Materials and Methods 1. Cell Viability AssayCell viability was observed by trypan blue staining. In briefly, after application of cyclic stretch, cells was harvested and stained with trypan blue for5min. To draw a small amount of cells from each sample and counted with a blood cell count board. Cell viability was calculated by the following formula: cell viability=(numbers of total cells-numbers of staining cells)/total cell numbers×100%.2. Cell culture and application of cyclic stretchHuman aortic smooth muscle cells (HASMCs) and Smooth Muscle Cell Medium (SMCM) were purchased from ScienCell. HASMCs were cultured in SMCM, which is designed for optimal growth of vascular smooth muscle cells, at37℃in an incubator with an atmosphere of5%CO2/95%air. For experiments, HASMCs (passages4to7) were plated onto6-well silicone elastomer coated with type I collagen (Flexcell). When reached confluent, cells were incubated in serum-free medium for an additional24h to achieve quiescence. On the day of the experiment, cells were replaced with fresh complete medium and then were subjected to cyclic stretch produced by the Flexercell Strain Unit FX-5000(Flexcell) for the indicated time at1Hz. Cells were divided into three groups as follows:(1) Static control:HASMCs were cultured in static conditions without mechanical stretch treatment.(2)10%elongation group:HASMCs were exposed to10%mechanical stretch for1h,3h,6h,12h, and24h, respectively;(3)15%elongation group:HASMCs were exposed to15%mechanical stretch for1h,3h,6h,12h, and24h, respectively;3. Real-time RT-PCRTotal RNA was isolated from HASMCs with Trizol (Invitrogen), lug total RNA was reverse-transcribed using PrimeScript(?) RT reagent Kit (Tarkara), following by one-step real-time PCR using SYBR Green technology on a Light Cycler (Bio-Rad). ACE2primers were:Forward5'-CATTGGAGCAAGTGTTGGATCTT-3', Reverse:5'-GAGCTAATGCATGCCATTCTCA-3'. ACE primers were Forward: 5'-GCGGCTCTTCCAGGAGCTGC-3', Reverse:5'-CTGCGCCCACATGTTCCCCA-3'; MAS primers were:Forward5'-GGCCTCCTCATGGATGGGTCAA-3', Reverse:5'-GTGCATTCCCGACTGAGGCGT-3'; The housekeeping gene β-actin was used as the internal control. The data were analyzed by the2-ΔΔCT method.4. Western blot analysisCells were harvested and proteins were extracted by using a cytoplasmic extraction reagents kit (Pierce). Protein extracts (20ug) were separated by SDS-PAGE, transferred to PVDF membrane (Millipore), blocked by5%nonfat milk for2hours and then membranes were incubated with primary antibodies against ACE2, and β-actin overnight at4℃. After washing with1×TBST for3times, membranes were incubated with a second antibody (1:10,000dilution) for1hour. Targeting proteins were visualized with the enhanced chemiluminescence plus detection system (Millipore). Relative band intensities were analyzed with Photoshop CS3software.5. ACE2activity assayACE2activity of HASMCs exposed to cyclic stretch for each time point was determined as described previously. In brief,20ug total protein extracts were incubated with7-Mca-YVADAPK (Dnp, luM), a substrate of ACE2, in a final volume of100μL reaction buffer at room temperature. Fluorescence kinetics was measured for4h by use of Varioskan Flash (Thermo Scientific, Worcester, MA, USA) at an excitation wavelength of320nm and an emission wavelength of400nm. EDTA (1mM) and recombinant human ACE2(25ng,(R&D Systems, USA) were used as negative and positive controls, respectively. ACE2activity was expressed as the difference in fluorescence with or without the ACE2inhibitor DX600(1uM, Belmont, CA, USA).6. Measurement of Ang II and Ang-(1-7) LevelsAfter stretch treatment for indicated time, cells were harvested and proteins were extracted by using a cytoplasmic extraction reagents kit (Pierce). By using a BCA protein assay kit, the protein concentration of each sample was measured. The Ang Ⅱ and Ang-(1—7) levels in HASMCs were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions.7. RNA interferenceHASMCs were transfected with interfering RNA (siRNA) of ACE2in different concentrations or negative control siRNA. The siRNA sequence for ACE2was as follows:sense:5'-CCA UCU ACA GUA CUG GAA A dTdT-3'; antisense:5'-UUU CCA GUA CUG UAG AUG G dTdT-3'.The negative control siRNA sequence was as flollows:sense:5'-UUC UCC GAA CGU GUC ACG U dtdt-3'; antisense:5'-ACG UGA CAC GUU CGG AGA A dtdt-3'. After incubation for24hours, cells were harvested and Western blot was performed to test the inhibitory effect of ACE2siRNA.8. Cell proliferation analysisCell proliferation analysis was performed by cell count and BrdU incorporation, respectively. After being exposed to cyclic stretch (10%elongation,1Hz) or under static conditions for12hours, HASMCs were washed with cold PBS and harvested by trypsinization and counted (Coulter). BrdU incorporation was performed according to the manufacturer's instructions (Roche Applied Science, Indianapolis, IN)9. Cell migrationScratch test was performed to evaluate the effect of cyclic stretch on HASMCs migration. Cells were plated directly onto silicone membranes of Flexcell6-well plates. When reached confluence, a line of cells was removed with a sterile100ul-pipette tip across the layer. The culture media was replaced with fresh complete media, and then the HASMCs were exposed to10%cyclic stretch or under static conditions for12hours. After that cells were fixed by incubation with90%ethanol for20min and the migrated distance was measured at10points along the wound edge by using the Photoshop CS3software.10. Animal protocolMice Transverse Aortic Constriction (TAC) model was performed to induce pressure overload. The operation protocols were approved by Institutional Animal Care and Use Committee. Male wild type C57mice weighing25-30g were anaesthetized with0.8%sodium pentobarbital. Transverse aortic constriction was performed between the left and right carotid arteries, and the sham-operated mice were used as negative controls, which undergoing the same procedures except for aortic constriction. After24hours, mice were killed with an overdose sodium pentobarbital and perfused with physiological saline. Bilateral carotid arteries were collected and the expression of ACE2was measured by RT-PCR, Western blot and immunofluorescence.11. immunofluorescenceHASMCs were exposed to cyclic stretch or under static conditions for12hours, and immunofluorescence was performed to further investigate the effects of stretch on ACE2protein expression according to the previous methods.In in-vivo experiment, serial cryostat sections were performed for immunofluorescence analysis of ACE2expression in the media of the vessel. Sections were blocked by5%BSA for1hour and then incubated with a rabbit monoclonal ACE2antibody (1:50; abcam) and a mouse monoclonal anti-smooth muscle cell actin antibody (1:100; Sigma-Aldrich) overnight together. For a negative control, the same amount of physiological saline was used instead of the primary ACE2antibody. After washing for3times with PBS, the cells were stained with Fluor secondary antibodies for1hour following with DAPI staining for10min. Images were gained using a confocal microscopy. The staining density was analyzed with Sigma Scan Pro.5.0.12. Statistical analysisAll data are presented as means±SEM. Results were from at least three independent experiments. Comparisons between groups and pairs were made by one-way ANOVA or Student's t-test, as appropriate. Statistical significance was defined as P<0.05.Results1. Stretch does not affect cell viabilityIn order to exclude the effect of cell injury induced by stretch on the expression of ACE2and its relevant biological roles, we performed trypan blue staining to monitor cell viability in HASMCs treated with stretch or not. The results revealed that cyclic stretch had no effect on cell viability compared with static control.2. Mechanical Stretch Modulates ACE2, ACE and MAS mRNA Expression in Vascular Smooth Muscle Cells in VitroThe mRNA expressions of ACE2, ACE and MAS in response to mechanical stretch were examined in HASMCs. Compared with static control, moderate mechanical stretch (10%elongation,1Hz) enhanced ACE2mRNA expression in cultured HASMCs with a peak at6h (3.1fold, P<0.01), and remained higher than control levels at24h. The MAS mRNA expression showed a very similar tendency to that of ACE2, which peaked at6h (2.4fold, P<0.01). The ACE mRNA expression slightly increased at first, then decreased at12h (0.41fold,P<0.05), which remained until at24h.When exposed to increased mechanical stretch (15%elongation,1Hz), both ACE2and MAS mRNA expression showed a delayed kinetic with a transient peak at3h (P<0.01and P<0.05, respectively), then declined at12h (0.37fold and0.44fold, respectively, P<0.05) and maintained at a reduced level at24h. Increased stretch (15%elongation,1Hz) induced a progressively elevated ACE mRNA expression with a peak at12h (2.35fold, P<0.01). In order to elucidate the different response of ACE2and ACE to the same stretch profile, a comparison between the mRNA expression of ACE2and ACE was made. Compared with ACE mRNA expression, ACE2presented a significantly higher mRNA expression under moderate stretch (10%elongation,1Hz)(P<0.01). However, increased stretch (15%elongation,1Hz) induced an enhanced mRNA expression of ACE as compared with that of ACE2(P<0.01).3. Mechanical Stretch Modulates ACE2protein Expression in Vascular Smooth Muscle Cells in VitroModerate mechanical stretch (10%elongation,1Hz) enhanced ACE2protein expression in cultured HASMCs at6h (P<0.01), and remained higher than that of static controls at24h (P<0.01). However, when exposed to increased mechanical stretch (15%elongation,1Hz), ACE2protein expression showed a delayed kinetic with a transient peak at3h (P<0.01) and then declined at12h and maintained a reduced trend after24hours of stretch (p<0.01).4. The Effect of Mechanical Stretch on ACE2Enzyme Activity in Vascular Smooth Muscle CellsModerate cyclic stretch (10%elongation,1Hz) enhanced ACE2Enzyme Activity in cultured HASMCs with a peak at12h (P<0.01), and remained higher than that of static controls at24h (P<0.01). However, when exposed to increased mechanical stretch (15%elongation,1Hz), ACE2enzyme activity first slightly increased and then declined at12h and remained at24h(p<0.01).5. Measurement of Ang Ⅱ and Ang-(1-7) LevelsModerate mechanical stretch (10%elongation,1Hz) increased Ang-(1-7) Levels and descreased Ang Ⅱ levels at12h in cultured HASMCs and the trend remained at24h (P<0.05, P<0.01). However, Ang-(1-7) levels decreased under increased mechanical stretch (15%elongation,1Hz) at12h and maintained at24h, while Ang Ⅱ levels increased at6h and remained at an increased level after24h of increased mechanical stretch(P<0.05, P<O.01).6. ACE2was involved in mechanical stretch induced proliferation suppression in HASMCs.To explore the role of ACE2induced by physiological moderate stretch (10%elongation,1Hz), we first investigated the effect of10%cyclic stretch on HASMCs proliferation though BrdU incorporation method and cell counting. After being stretched for12hours, BrdU-positive cells decreased3.8-fold compared with static control, which indicated that10%stretch suppressed HASMCs proliferation. Next, HASMCs were transfected with ACE2siRNA or negative control siRNA, and then exposed to10%stretch for12hours, BrdU-positive cells increased2.1-fold compared with stretched cells with scramble siRNA. Results from cell counting method were in agreement with BrdU incorporation assay, which indicated that the ACE2was involved in10%stretch mediated proliferation suppression effect. 7. ACE2participated in stretch-induced HASMCs migration suppressionCell scratch test demonstrated that the migration distance of HASMCs was inhibited significantly after12hours of cyclic stretch (10%elongation,1Hz) as compared with that of static control. The suppression effect of stretch on HASMCs migration was partly abolished by ACE2knockdown with siRNA.8. Pressure Overload Reduces ACE2Expression in VivoACE2mRNA expression decreased in aortic segments proximal to the coarctation (pressure overload) as compared with that distal to the constriction and in sham controls. The ACE2protein expression was also found to be suppressed by enhanced stretch. OCT-embedded aortic segments of pressure overload presented a down-regulated ACE2expression in the medial layer of aorta, verified by a-SMC staining,24h after TAC as compared with that in the segments of low pressure (distal to the constriction) and sham controls.Conclusions1. Mechanical stretch can modulate the expression of ACE2in vascular smooth muscle cells.2. ACE2plays a key role in the suppression effect of moderate cyclic stretch on proliferation and migration in HASMCs.3. Pressure overload reduces ACE2expression in vivo. BackgroundAlthough pharmacologic strategies for cardiovascular (CV) events and related end-organ damage have made great progress, CV disease is still the major cause of death in the world. It has been generally accepted that vascular remodeling is an important feature in vascular pathology. Thus, novel therapeutic agents aimed at reducing vascular remodeling must be developed for preventing and reducing residual CV events. As a target tissue of hypertension, arterial blood vessels are constantly exposed to mechanical forces in the form of stretch as the result of the pulsatile nature of circulating blood. Blood pressure, which is the major determinant of vessel stretch, can create radial forces that counteract the intraluminal pressure and affect all cell types in the vessel. Hypertension is closely associated with the structural remodeling of vascular wall. Both mechanical stretch and neurohumoral factors play key roles in promoting vascular remodeling via excessive activation of renin-angiotensin system (RAS).The renin-angiotensin system (RAS), an important candidate as a causative factor in the development and maintenance of hypertension, has been extensively studied since renin was discovered as a pressor substance in1898. This endocrine cascade convert the inactive prohormone Ang I to the active peptide Ang II, which is vasoconstrictive, and plays a key role in renal sodium and water absorption by binding to the AT1receptor. Initially, it was thought that its effect on blood pressure is mediated mainly by the classical endocrine pathway; that is, the blood-borne Ang Ⅱ acts on target tissues. More recently, however, it has been accepted that local RAS exist and are physiologically active in an autocrine or paracrine pathway in many tissues. The recently discovered angiotensin converting enzyme-2(ACE2) and the MAS receptor has made a renewed emphasis on understanding the role of original cascade and led to the emergence of a new axis composed of ACE2/Ang-(1-7)/MAS, which is provasodilatory, antifibrotic and antigrowth. Accordingly, the new axis is now seen as a negative regulator of ACE/AngII/AT1receptor axis.Cyclic stretch due to the heart beats plays key roles for the maintenance of vascular structure and function. The smooth muscle cells located in the medial layer of the vessel wall are the primary target cells for cyclic stretch. Stretch-induced function disorders of smooth muscle cells are implicated in vascular pathological remodeling. As a recently discovered RAS member, ACE2has a wide range of cardiovascular and renal protective effects. Studies confirmed that other members of RAS, such as ACE and AT1R, were involved in stretch mediated biological effects. Previously we have already observed the effects of stretch on ACE2expression in smooth muscle cells. The mechanisms underlying the regulation of ACE2expression, however, are still poorly understood. The present study will focus on elucidating the molecular mechanisms of ACE2expression mediated by stretch, which is of great importance for further clarifying the molecular mechanisms of vascular remodeling and providing new ideas and targets for clinical intervention.Objectives1. To verify the transcription factors involved in mechanical stretch-induced ACE2expression.2. To elucidate the role of MAPK pathway in mechanical stretch-induced ACE2expression.3. To investigate the effect of PKC-(βⅡin mechanical stretch-induced ACE2expression. 4. To identify the role of Akt in mechanical stretch-induced ACE2expression.Methods1. Cell culture and application of cyclic stretchHuman aortic smooth muscle cells (HASMCs) and Smooth Muscle Cell Medium (SMCM) were purchased from ScienCell. HASMCs were cultured in SMCM, a complete medium designed for optimal growth of vascular smooth muscle cells, at37℃in an incubator with an atmosphere of5%CO2/95%air. For experiments, HASMCs (passages4to7) were plated onto6-well silicone elastomer coated with type I collagen (Flexcell). When reached90%confluent, cells were incubated in serum-free medium for an additional24hours to achieve quiescence. On the day of the experiment, cells were replaced with fresh complete medium and then subjected to cyclic stretch produced by the Flexcell-5000Tension Unit for the indicated time at1Hz. For inhibiting the intracellular signaling pathways, specific pharmacological inhibitors were added to the media of HASMCs1hour before stretch stimulation.2. RNA stabilityAfter being subjected to cyclic stretch for12hours, transcription inhibitor actinomycin D (5μg/ml) was added to the media of HASMCs with or without stretch. ACE2mRNA levels were analyzed by qRT-PCR at indicated time points. β-actin mRNA levels were also measured as the internal control. The results were expressed as the ratio of mRNA levels at each time point as compared to those at the time of initial actinomycin D treatment (0h).3. Promoter activity assayA human ACE2promoter construct was generated. A2000bp fragment of human ACE2promoter region (full length,-1908to+1; GenBank accession number:13557) was subcloned into the pGL3-basic vector by PCR using Pfu polymerase. The ACE2promoter construct and PRL-TK vector were transiently cotransfected into HASMCs by lipofectamine2000according to the manufactures'protocol for24hours before being treated with cyclic stretch. After being stretched for3hours, the extracts of both the static and the stretched cells were prepared and ACE2promoter activity was measured by using Dual-Luciferase Reporter Assay System (Promega).4. Experimental groups for verifying the transcription factors involved in mechanical-stretch induced ACE2expression1) Static control:HASMCs cultured in static conditions without any inhibitors;2)10%elongation group:HASMCs were exposed to10%mechanical stretch for15min,30min,1h2h, respectively;3)10%stretch following treatment of pharmacological inhibitors:HASMCs were exposed to10%mechanical stretch for6h following1h pretreatment with SN50(18μM), Curcumin(1OuM) and WP631(100nM) respectively.Then to detect the protein expression of p65, p-p65, c-fos, p-c-fos, c-jun, p-c-jun, Sp-1, p-Sp-1and ACE2by Western blot, the ACE2mRNA expression by RT-PCR, and the DNA binding ability of NF-κB and AP-lby EMS A.5. Investigating the effect of PKC-βⅡ in mechanical stretch induced ACE2expression1) Static control:HASMCs cultured in static conditions without any inhibitors;2)10%elongation group:HASMCs were exposed to10%mechanical stretch for15min,30min,1h,2h, respectively;3)10%stretch following pretreatment with pharmacological inhibitor of PKC-βⅡ: HASMCs were pretreated with CG53353(100nM)1hours and exposed to10%mechanical stretch for6hours;Then the protein expression of PKC-βⅡ, p-PKC-βⅡ and ACE2was detected by Western blot, and the ACE2mRNA expression was detected by RT-PCR. The DNA binding ability of NF-κB and AP-1was detected by EMS A.6. Identifying the role of Akt pathway in mechanical stretch-induced ACE2expression1) Static control:HASMCs cultured in static conditions without any inhibitors;2)10%elongation group:HASMCs were exposed to10%mechanical stretch for15mi,30min,1h,2h, respectively;3)10%stretch following pretreatment with pharmacological inhibitors: HASMCs were exposed to10%mechanical stretch for6h following1h pretreatment with LY294002(50μM); Then the protein expression of Akt, p-Akt and ACE2was detected by Western blot and the ACE2mRNA expression was detected by qRT-PCR.7. Elucidating the role of MAPK pathway in mechanical-stretch induced ACE2expression1) Static control:HASMCs cultured in static conditions without any inhibitors;2)10%elongation group:HASMCs were exposed to10%mechanical stretch for15min,30min,1h,2h, respectively;3)10%stretch following treatment with pharmacological inhibitors:HASMCs were exposed to10%mechanical stretch for6h following1hours pretreatment with PD98059(30μM), SP600125(20μM) and SB203580(10μM) respectively.Then to observe the protein expression of ERK1/2, p-ERK1/2, p38, p-p38, JNK1/2, p-JNK1/2and ACE2by Western blot, the ACE2mRNA expression by RT-PCR, and the binding ability of NF-κB and AP-1by EMSA.8. Western blotAfter treatment with cyclic stretch for indicated time, cells were harvested and proteins were extracted by using a cytoplasmic extraction reagents kit (Pierce). Protein extracts (20ug) were separated by SDS-PAGE, transferred to PVDF membrane (Millipore), blocked by5%nonfat milk for2hours and then membranes were incubated with primary antibodies against ACE2, PKCβⅡ, p-PKCβⅡ, Akt p-Akt, JNK1/2, p-JNK1/2, p38, p-p38, ERK1/2, p-ERK1/2, p65, p-p65, c-jun, p-c-jun, c-fos, p-c-fos, Sp-1, p-Sp-1and β-actin overnight at4℃. After washing with1×TBST for3times, membranes were incubated with a second antibody (1:10,000dilution) for1hour. Targeting proteins were visualized with the enhanced chemiluminescence plus detection system (Millipore). Relative band intensities were analyzed with Photoshop CS3software.9. Real-time RT-PCRTotal RNA was isolated from HASMCs exposed to cyclic stretch for indicated time by using Trizol (Invitrogen), lug total RNA was reverse-transcribed using PrimeScript(?) RT reagent Kit (Takara), following by one-step real-time PCR using SYBR Green technology on a Light Cycler (Bio-Rad). ACE2primers were Forward:5'-CATTGGAGCAAGTGTTGGATCTT-3', Reverse:5'-GAGCTAATGCATGCCATTCTCA-3'. The housekeeping gene β-actin was used as the internal control. The data was analyzed by the2-ΔΔCT method.10. Electrophoretic Mobility Shift Assay (EMSA)Nuclear protein was extracted from HASMCs by using nuclear protein extraction kit (Pierce), and the protein concentrations were determined by BCA assay method. The sequence of double-stranded gel shift oligonucleotides for AP-1and NF-κB are:5'-CGC TTG ATG ACT CAG CCG GAA-3'and5'-AGT TGA GGG GAC TTT CCC AGG C-3'respectively, which was end-labeled with biotin. The nuclear extracts (10ug) were mixed with labeled oligonucleotides for AP-1or NF-κB and other important components in a total volume of20ul for20min, and then separated by6%nondenaturing acrylamide PAGE. The following procedures were according to the manufactures'instructions.11. Statistical analysisAll data are presented as means±SEM. Results were from at least three independent experiments. Comparison between groups was made by one-way ANOVA and Student's t test was used to analyze paired samples. Statistical significance was defined as P<0.05.Results1. stretch-induced ACE2expression was not due to decreasing the ACE2mRNA degradationIn order to investigate whether stretch-induced ACE2expression was caused by decreasing the ACE2mRNA degradation, HASMCs were treated with a transcription inhibitor actinomyctn D following with or without stretch for12h. qRT-PCR was used to determine the ACE2mRNA levels at the time points of0,6,12hours from the addition of actinomycin D, respectively. As shown in fig.1, the ACE2mRNA levels in cells with or without stretch treatment decayed at comparable rates, demonstrating that stretch may not influence the ACE2mRNA stability in HASMCs.2.10%cyclic stretch enhanced ACE2promoter activityFull-length of ACE2promoter was cloned into the luciferase reporter vector pGL3-basic to generate pGL3-ACE2-promoter vector. Cultured HASMCs were transiently cotransfected with ACE2promoter vector and PRL-TK vector, and then exposed to10%stretch for3hours. DLR assay was performed to determine the ACE2promoter activity in stretched or static control cells. As shown in fig.2,10%cyclic stretch significantly increased ACE2promoter activity in HASMCs as compared with static control. This indicated that cyclic stretch could regulate ACE2expression at transcription level.3. NF-κB may suppress mechanical stretch-induced ACE2expression in HASMCsData from Western blot indicated that p-p65levels were higher in stretched cells than that in static control. Addition of SN50(a specific inhibitor for NF-κB) further enhanced ACE2expression in HASMCs induced by stretch both in protein and mRNA level. Gel shift experiment proved that stretch significantly increased the DNA binding activity of NF-κB in a time-dependent way, which peaked at3hour after stretch. A100-fold excessive of cold competitive NF-κB oligonucleotides eliminated the DNA/protein complex binding. These experiments revealed that the increased NF-κB binding activity may be a negative regulator of ACE2expression in HASMCs undergoing stretch treatment.4. Enhanced AP-1activity was essential for mechanical stretch-induced ACE2expression in HASMCsData from Western blot indicated that stretch significantly enhanced the phosphorylation of c-jun, and immunofluorescence also revealed an increased c-jun level in the nuclear region after stretch. Addition of curcumin (a potent inhibitor for AP-1activity) to the media of cultured HASMCs1hour prior to stretch significantly abolished stretch-induced ACE2protein and mRNA expression. Gel shift experiment revealed that stretch significantly increased the DNA binding activity of AP-1in a time-dependent way, which peaked at3hour. A100-fold excessive of cold competitive AP-1oligonucleotides eliminated the DNA/protein complex binding. These experiments revealed that stretch-induced ACE2up-regulation could be AP-1dependent, c-jun but not c-fos may play a key role in the process.5. Sp-1may not participate in mechanical stretch-induced ACE2expression in HASMCsAlthough stretch significantly enhanced the phosphorylation of Sp-1, WP631, a pharmacological inhibitor of Sp-1, did not affect ACE2mRNA and protein expression in the presence of stretch treatment, which means Sp-1doesn't participate in the mechanical-stretch induced expression of ACE2in HASMCs.6. JNKl/2is partially involved in mechanical stretch-induced ACE2expression in a NF-κB/AP-1-dependent manner in HASMCsAfter mechanical stretch treatment, JNK1/2pathways were activated, demonstrating by their phosphorylation level. Phosphorylation of JNK1/2increased at15min and peaked at30min. Pharmacological inhibitors for JNK1/2were added to the media of cultured HASMCs1hour before stretch intervention, and then the HASMCs were exposed to10%cyclic stretch for6hours. As shown in fig15, SP600125(inhibitor of JNK1/2) significantly attenuated stretch-induced ACE2protein and mRNA expression. To test whether JNK1/2regulate stretch-induced ACE2expression though an AP-1or NF-κB DNA dependent way, cells pretreated with SP600125were stretched by10%elongation for3hours, Gel shift experiment demonstrated that SP600125attenuated stretch-induced AP-1and NF-κB DNA binding activity. These results indicated that JNK1/2signal pathway may play an important role in regulating ACE2expression mediated by10%stretch.7. PKC-βⅡ plays a key role in mechanical stretch-induced ACE2expression in a NF-κB/AP-1-dependent manner in HASMCsAfter mechanical stretch treatment, a rapid activation of PKC-βⅡoccurred in HASMCs, which was determined by its phosphorylation levels. Phosphorylation of PKC-βⅡ increased at15min and peaked at30min. GC53353, an inhibitor specific for PKC-βⅡ, significantly attenuated stretch-induced ACE2protein and mRNA expression. To test whether PKCβⅡ regulate stretch-induced ACE2expression in an AP-1or NF-κB dependent way, cells pretreated with GC53353were stretched by10%elongation for3hours, Gel shift experiment revealed that GC53353attenuated stretch-induced AP-1and NF-κB DNA binding activity. These results indicated that PKC-βⅡsignal pathway may play an important role in10%stretch-induced ACE2expression.8. ERK1/2, p38MAPK and Akt pathways are not involved in mechanical stretch-induced ACE2expression in HASMCsAlthough mechanical stretch could trigger a rapid activation of ERK1/2, p38MAPK and Akt pathways, which was demonstrated by increased phosphorylation levels. Pretreatment with PD98059(inhibitor of ERK1/2), SB203580(inhibitor specific for p38) and LY294002(inhibitor of Akt) had no effec...
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