| The role of ACE2 in pulsatile shear stress-mediated functions of vascular endothelial cellsBackgroundVascular diseases including coronary disease, ischemic stroke et.al, have become the major burden for our health. Atherosclerosis (AS) is the main pathological basis of these diseases. The mechanism for the development of AS was not complete clear yet, recently, more and more studies focus on the relationship of the mechanical forces and AS.The blood vessels were constantly exposed to mechanical stretch and shear stress due to heat beat and circulating blood, respectively. Endothelial cells (ECs) dysfunction was the first step for the development of AS, and many studies had indicated that shear stress mainly mediated ECs biological functions, such as proliferation, the progression of cell cycle, migration, et al. According to previous studies, shear stress was sensed by endothelium and transduced into intercellular chemical signals by some receptors or ion channels on ECs surface, activating some signal pathways, promoting or suppressing some genes expression related to ECs functions. In recent years, more and more genes were proved to be involved in shear stress-mediated ECs and vascular function.Shear stress was diverse in different regions of blood vessels. In the straight part of the vessel, the blood flow was uniform and the ECs undergo pulsatile laminar shear stress (PSS), however, in vessels of bend or bifurcation where blood flow was disturbed, the ECs suffered low shear stress (LSS) or oscillatory shear stress (OSS). Ample evidence indicate PSS play a important role maintaining ECs function, while LSS or OSS induce ECs display a pro-atherosclerosis phenotype.Over activation of renin-angiotensin system (RAS) was closely related with the development of AS, and AngⅡ was the main effect factor. AngⅡ could promote ECs express many inflammatory factors, inducing endothelium dysfunction. Inhibition the role of AngⅡ has become one of the basic strategies for AS treatment. Angiotension-converting enzyme 2 (ACE2), the homologue of ACE, could degrade AngⅡ into Ang-(1-7), which has cardiovascular protective effects. Recently, many in vivo and in vitro studies revealed ACE2 play a vital role for maintaining endothelial homeostasis, exerted significant anti-atherosclerosis effect. Previous studies showed that shear stress regulated ACE expression in ECs, and AngⅡ level was also altered by shear stress. However, the effect of shear stress on ACE2 expression is still unclear.ACE2 expression is abundant in ECs. Results of several recent studies indicated its strongly protective roles of endothelium, and shear stress is also an important factor for functions of ECs, thus, we put forward a hypothesis that ACE2 expression is likely affected by shear stress, participating in regulation of ECs functions. To make clear the effect of shear stress on ACE2 expression will help us further understand the relationship between RAS and AS.Objectives1. To explore the effect of pulsatile shear stress (PSS) on ACE2 expression in cultured HUVECs.2. To investigate the role of ACE2 in PSS-mediated biological functions of ECs in vitro.3. To investigate the effect of shear stress on ECs ACE2 expression in vivo.Materials and methods1. Cell CultureHuman umbilical vein endothelial cells (HUVECs) were isolated from fresh human umbilical cords by trypsin digestion under sterile conditions. The cells was re-suspended in culture medium containing M199 medium,20% (vol/vol) fetal bovine serum (FBS),2 ng/ml fibroblast growth factor-2, and 1% (vol/vol) penicillin/streptomycin. And then cultured in an incubator with an atmosphere of 5% CO2 at 37℃. The cell identification was performed using CD31 immunofluorescence staining before experiment.The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki with the approval of the Institutional Medical Ethics Committee of Qilu Hospital, Shandong University.2. Application of shear stressPassage 4-8 cells were used in experiment. HUVECs were plated onto collagen I coated slides. When cells reached into 90% confluence, they were exposed to different patterns of shear stress for indicated time by use of a Flexcell streamer, which is controlled by a computer. First, in order to investigate the effect of PSS on ACE2 expression in vitro, the cells were subjected to pulsatile shear stress (PSS,12 ±4dyn/cm2, 1Hz) for indicated times (0,1,3,6,12,18hr), the ACE2 expression level was determined by qRT-PCR and WB; besides, in order to evaluate different patterns of shear stress on ACE2 expression, the cultured HUVECs were exposed to oscillatory shear stress (OSS,0±4 dyn/cm2, 1Hz), PSS and static condition for 12 hr, respectively. After experiment, the qRT-PCR, WB and Immunofluorescence were used to investigate ACE2 expression level.3. Real time quantitative-PCR (qRT-PCR)Total RNA was extracted by use of Trizol (Invitrogen) and then transcribed into cDNA using a cDNA synthesis kit (TAKARA). qRT-PCR was performed by use of TAKARA SYBR Green Mix kit according to manufacturers’ protocol. Primers for ACE2 and GAPDH were synthesized by Shanghai GenePharm.4. Western blot analysisHUVECs being sheared or maintained at static conditions were harvested and lysed using protein lysis buffer, the protein was extracted and concentration was determined by BCA method.20ug protein was separated by SDS-PAGE, and transferred into PDVF membrane, following with blocking, incubating with primary and second antibodies, washing. Visualization was performed by using an enhanced chemiluminscence-plus detection system (Millipore). β-actin was considered as an internal reference.5. ImmunofluorescenceAfter experiment cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% TritonX-100. After incubation overnight at 4℃ with anti-ACE2 antibodies, cells were incubated for 1 hour with Alexa 647-conjugated secondary antibodies. Nuclei were stained with DAPI. Cells were investigated by use of a fluorescent microscope.6. Detecting of AngⅡ and Ang-(1-7) levelCells were harvested and protein was extracted, after detecting the protein concentration with BCA method. The AngⅡ and Ang-(1-7) level in cells under different conditions were determined by using of ELISA method according to the manufactures’ instructions.7. Analysis of NO levelHUVECs were harvested and lysed by protein lysis buffer, after determining the protein concentration, NO level in cytoplasm was detecting by use of commercial kit gained from Beyotime Biotechnology according to the manufacturer’s instructions.8. Transient TransfectionFor knockdown ACE2 expression, specific siRNA for human ACE2 was designed and synthesized by Shanghai GenePharm. When cells grown into 80% confluence, ACE2 siRNA was transfected into cells using Lipofectamine 2000 (Invitrogen). The effect of ACE2 siRNA was detected by Western blot.9.BrdU incorporation assayThe effect of ACE2 in PSS-mediated proliferation of HUVECs was detected by BrdU incorporation method. The detailed protocols were according with the manufactures’ instructions. At last, the BrdU-positive cells were investigated by a laser-scanning microscope and counted.lO.Apoptosis detection by TUNELThe apoptosis of HUVECs was detected by use of Apoptag Plus Peroxidase In Situ apoptosis detection kit, The detailed protocols were according with the manufactures’ instructions.11. En Face StainingFive 10-12 week C57/BL6 mice were gained from Shandong university, All animal experiments were performed in accordance with the Animal Management Rules of the Chinese Ministry of Healthand with the approval of the Animal Care Committee of Shandong University.Mice were anesthetized with 0.8% (wt/vol) pentobarbital sodium, following by perfused with 30 ml lukewarm saline,80 mL of 4% paraformaldehyde. After perfusion, aortas were gained and post-fixed in the fixed solution for 4 hr and then subjected to en face immunostaining. In brief, the adventitia was removed carefully, aortas were longitudinally dissected, blocked with 5 (vol/vol) BSA, then incubated with specific primary antibodies rat anti-CD31 mAb and rabbit anti-ACE2 mAb at 4℃ overnight, then FITC-conjugated anti-rat IgG and Alexa Fluor 647-conjugated anti-rabbit IgG second antibodies. Samples were counterstained with DAPI for nuclei, photographed under a confocal microscope.12. Statistical AnalysisAll data were shown as mean±SEM. Statistical analysis method involved independent Student’t test for 2 groups and one-way ANOVA for multiple comparisons.p<0.05 was considered statistically significant.Results1. Pulsatile shear stress (PSS) upregulated ACE2 expression in cultured HUVECsFirst, cultured HUVECs were subjected to PSS for 0,1,3,6,12,18 hr, respectively, and qRT-PCR and WB were performed to evaluate the ACE2 expression. The results revealed that Compared to static control (0 hr), PSS significantly increased ACE2 mRNA at 3 hr, untile 18 hr (p<0.05), the ACE2 protein level began to increase at 6 hr untile 18 hr, the peak at 12 hr (p<0.05).In addition, in order to evaluate different patterns of shear stress on ACE2 expression in ECs. The cultured HUVECs were exposed to OSS, PSS or were maintained at static condition. WB, qRT-PCR and Immunofluorescence were used to determine the ACE2 expression, the experimental data indicated that OSS also increased ACE2 expression both at mRNA and protein level (p<0.05); however, compared to PSS, the ACE2 expression level in ECs sheared by OSS was decreased significantly (p<0.05).Besides, we also observed that HUVECs sheared by 12 hrs PSS were elongated and aligned with longitudinal axis of the fluid flow, however, cells undergone OSS or maintained at static conditions showed a polygonal morphology without obvious polarity.2. PSS increased Ang-(1-7) and reduced AngⅡ level in HUVECsCultured HUVECs were exposed to PSS or maintained at static control for 12hrs, the Ang-(1-7) and AngⅡ level in cytoplasm was determined by ELISA method. the experimental data showed that PSS significantly increased Ang-(1-7) level and reduced AngⅡ level compared to static control (p<0.05).3. ACE2 was involved in PSS mediated NO production in cultured HUVECs.After 12 hrs PSS stimuli, cells were collected for detecting the level of NO production. Compared to static control, PSS obviously increased NO production (p<0.05); inhibition of ACE2 expression by siRNA, the up-regulation of NO level by PSS was partially abolished. In order to investigate the mechanisms of ACE2 involving in PSS-induced NO production, we detected eNOS and p-eNOS level by Western blot, results indicated that PSS induced eNOS, p-eNOS expression were partially inhibited by ACE2 siRNA (p<0.05). In addition, the Ang-(1-7) and AngⅡ levels in HUVECs cytoplasm of each groups were also investigated by ELISA method, the experimental data indicated that transfection of ACE2 siRNA significantly reduced Ang-(1-7) level, but increased AngⅡ level in sheared cells compared with that of transfection of NC siRNA (p<0.05).4. ACE2 was involved in inhibition of VCAM-1, MCP-1 expression by PSS.Cultured HUVECs were pretreated with TNF-α (100nM) for 12 hrs, then subjected to PSS or maintained at static condition for 12 hrs. Results of WB demonstrated that the VCAM-1 and MCP-1 protein expression was decreased significantly compared to static control (p<0.05), however, knockdown of ACE2 expression by siRNA, PSS induced reduction of VCAM-1, MCP-1 expression was partially abolished as compared with NC treatment (p<0.05).5. The expression of ACE2 was higher in straight segment of thoracic aortas than that of inner curvature aortic arches.Endothelial cells of straight segment of thoracic aortas were exposed to pulsatile shear stress (PSS), however, endothelial cells of inner curvature aortic arches were mainly undergone low or oscillatory shear stress (OSS) stimuli. By use of en face staining, we found that the level of ACE2 in straight segment of thoracic aortas was significantly higher compared to inner curvature aortic arches. Thus, this result indicated PSS maintained a higher level of ACE2 in endothelial cells, playing an important anti-AS role.Conclusion(1) Pulsatile shear stress (PSS) significantly enhanced ACE2 expression in vascular endothelial cells(2) ACE2 was involved in PSS-induced NO production in endothelial cells(3) ACE2 was participated in PSS- reduced VCAM-1 and MCP-1 expression in endothelial cells.The molecular mechanism of pulsatile shear stress-induced ACE2 expression in cultured HUVECsBackgroundThe vascular endothelial cells are constantly subjected to shear stress. The pulsatile shear stress plays an important role in maintaining normal functions of endothelium, such as anti-inflammation, anti-atherosclerosis; however, LSS or OSS due to disturbed flow induced more ROS production and reduced NO level significantly. Atherosclerosis is prone in these areas with disturbed flow. Recently, more and more shear stress sensitive genes were found to be involved in this process.ACE2 has powerful endothelial protective effect and anti-AS effect. Our previous results found PSS significantly induced ACE2 expression in cultured HUVECs, but we still have limited knowledge about the mechanism of ACE2 expression regulation. Shear stress could regulate gene expression at different level, including transcriptional, post-transcriptional, post-translation level. Studies revealed that PSS reduced ACE expression at transcriptional and post- transcriptional level. Shear stress regulated NO production also occurs at multiple levels. But the mechanism of up-regulation of ACE2 expression by PSS was unclear.AMP-activated protein kinase (AMPK) was a Ser/Thr protein kinase which was a key kinase for PSS mediated endothelial protective effect. PSS can induce the activation of the AMPK pathway to promote NO production and decrease ROS synthesis in endothelial cells; shear stress upregulated endothelial cell miR-143/145 leve through activation of the AMPK pathway, the latter participated in inhibiting the expression of ACE at post transcriptional level; a recent study shows that, in hypoxia conditions, AMPK agonists AICAR can up-regulate the expression of Sirtl, promoting ACE2 transcription. Thus, there is a close relationship between AMPK and RAS, yet whether AMPK is involved in the regulation of ACE2 by PSS? This problem needs further investigation.Kru"ppel-Like Factor 2 (KLF2) is a key transcription factor involved in PSS mediated biological functions of ECs. The study confirmed that, PSS enhanced KLF2 level though the activation of AMPK-ERK5/MEF2 pathway, and KLF2 was responsible for about 50-70% PSS induced gene expression. It has obviously anti-inflammatory, antioxidation functions. In various conditions, the relationship between ACE2 and KLF2 relationship is not clear, but given the endothelial protective effects of the two, we hypothesized that KLF2 may be an important regulator of the expression of ACE2 induced by PSS. AMPK pathway is an important upstream signaling pathway of KLF2, therefore, we speculate that pulstaile shear stress may up-regulate ACE2 expression via AMPK-KLF2 pathwayObjectives(1) To investigate the effect of PSS on ACE2 mRNA stability and promoter activity.(2) To investigate if AMPK-KLF2 pathwaywas involved in PSS induced ACE2 expressionMethods1. Cell culture and shear stress applicationTo recover the frozen passage 1 HUVECs, passage 4-7 was used for experiment. When grow into 80-90% confluence in complete medium(M199、10%FBS、100U/ml Penicillin-Streptomycin、2ng/ml growth factor), cells were exposed to PSS(12±4dyn/cm2, 1Hz) for indicated time. PSS was generated by Flexcell-5000 streamer which was controlled by a computer.2.ACE2 mRNA stabilityHUVECs were incubated with transcription inhibitor actinomycin D (5μg/ml) for 30 min, and then were exposed to PSS treatment or were kept in static condition for indicated time. ACE2 mRNA levels were analyzed by qRT-PCR, and β-actin mRNA levels were also measured as the internal control. The results were shown as the ratio of mRNA levels at each time point compared with those at the time of initial shear stress treatment (0 h).3. Promoter activity assayA human ACE2 promoter construct was designed and generated by Shanghai GenePharm. A 2000 bp fragment of human ACE2 promoter region (full length,-1908 to+1; GenBank accession number:13557) was subcloned into the pGL3-basic vector by PCR using Pfu polymerase. The ACE2 promoter construct and PRL-TK vector were transiently co-transfected into HUVECs using lipo 2000 according to the manufactures’protocol for 24 hours before PSS stimuli. After being sheared for 3 hours, the extracts of both the static and the sheared cells were prepared and the ACE2 promoter activity was measured by using Dual-Luciferase Reporter Assay System (Promega).4. Western blot analysisHUVECs being sheared or maintained at static conditions were harvested and lysed using protein lysis buffer, the protein was extracted and concentration was determined by BCA method.20ug protein was separated by SDS-PAGE, and transferred into PDVF membrane, following with blocking, incubating with primary and second antibodies, washing. Visualization was performed by using an enhanced chemiluminscence-plus detection system (Millipore). β -actin was considered as an internal reference.5. real time quantitative-PCRTotal RNA was extracted by use of Trizol (Invitrogen) and transcribed into cDNA using a cDNA synthesis kit (TAKARA). Then real time PCR was performed by use of TAKARA SYBR Green Mix kit according to manufacturers’ protocol. Primers for ACE2 and GAPDH were synthesized by Shanghai GenePharm.6. Statistical AnalysisAll data were shown as mean±SEM. Statistical analysis method involved independent Student’ t test for 2 groups and one-way ANOVA for multiple comparisons. P<0.05 was considered statistically significant.Results1. PSS did not affect ACE2 mRNA stabilityCompared to static controls, the degree of ACE2 mRNA stability in sheared HUVECs was not affected at each time point.2. PSS significantly enhanced ACE2 promoter activityHuman ACE2 promoter was cloned into the luciferase reporter vector pGL3- basic to construct pGL3-ACE2-promoter vector. HUVECs were cotransfected with ACE2 promoter and PRL-TK vector, and then subjected to PSS for 3 hrs. DLR assay was performed to determine the ACE2 promoter activity in sheared or static control cells. Results showed PSS significantly increased ACE2 promoter activity compared with static control. This indicated that PSS could promote ACE2 expression at transcriptional level.3. KLF2 was involved in up-regulation of ACE2 by PSSThe cultured HUVECs were subjected to PSS for 0.5,1,3,6 hr, then cells were collected for detecting KLF2 expression. WB results showed KLF2 expression began to increase at 1 hr (p<0.05); inhibition of KLF2 expression by siRNA, the PSS induced up-regulation of ACE2 expression was partially reduced compared to negative control (p<0.05).4. Akt pathway was not responsible for PSS-induced ACE2 expressionTo test PSS-induced Akt pathway activation, HUVECs was exposed to PSS for 0, 0.5,1,3,6hrs, respectively. The results of WB showed that p-Akt/total-Akt level increased significantly at 0.5hr, and last for 1hr. To explore whether Akt pathway was involved in PSS-induced ACE2 expression, its specific inhibitor LY294002 was added to the medium 30min before PSS stimuli, our results indicated that LY294002 has no effect on PSS-induced ACE2 expression compared to DMSO, these data demonstrated Akt pathway was not responsible for PSS-induced ACE2 expression.5. AMPK pathway was responsible for PSS induced ACE2 expressionThe cultured HUVECs were subjected to PSS for 0,0.5,1,3,6hrs, respectively. WB results indicated p-AMPKa2 level increased significantly at 0.5 hr(p<0.05)-, pretreated HUVECs with AMPK inhibitor Compound C(100uM) for 1hr, then cells were given PSS stimuli for different time points, the PSS induced KLF2 and ACE2 expression was significant reduced(p<0.05). These results indicated AMPK-KLF2 pathway was involved in PSS induced ACE2 expression.Conclusion(1) PSS regulates ACE2 expression at transcriptional level(2) PSS increased ACE2 expression via AMPK-KLF2 pathway.(3) Akt pathway was not involved in PSS induced ACE2 expression. |
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