| BackgroundAtherosclerotic plaques preferentially develop at arterial branches and curvatures where endothelial cells bear low and oscillatory shear stress induced by disturbed blood flow, as opposed to straight parts which feature protective unidirectional pulsatile shear stress. Vascular endothelial cells, a monolayer in direct contact with the flowing blood, bear the most of the wall shear stresses and play an important role in maintaining homeostasis in response to stress. Ample evidence shows that pro-atherosclerotic disturbed flow induces sustained activation of atherogenic genes in endothelial cells to promote their oxidation, inflammation, cell cycle progression and proliferation, whereas pulsatile shear stress tends to maintain endothelial cells in a quiescent and less proliferative state with a low level of oxidation and inflammation.Programmed cell death4(PDCD4) is an important tumor suppressor in the development of various human cancers and inhibits translation rather than transcription. Specifically, the PDCD4protein combines directly with the mRNA coding region of the target gene (MYB/c-MYB) to block translation. It can also compete with eukaryotic translation initiation factor (eIF)4G and ribonucleic acid (RNA) for eIF4A binding and trap eIF4A in an inactive conformation to inhibit translation initiation via its two highly conserved MA3domains. Thus, PDCD4regulates molecules functioning during tumor cell proliferation, apoptosis, transformation, invasion and autophagy. Although PDCD4in general suppresses the development and progression of tumors, its specific biological functions differ by cell type.PDCD4also plays a role in cardiovascular cell biology by inhibiting proliferation and inducing apoptosis of most cardiovascular cells, including vascular smooth muscle cells, cardiac myocytes and fibroblasts, and repressing contractile gene expression in vascular smooth muscle cells. However, little is known about the action of PDCD4in endothelial cells.Our recent study of PDCD4and ApoE double-deficient mice (PDCD4-/-ApoE-/-) fed a high-cholesterol diet found that knockout of PDCD4was associated with reduced atherosclerosis plaque areas, so PDCD4downregulation might protect arteries against atherosclerosis. To determine whether PDCD4plays a role in the response of vascular endothelial cells to shear stresses, we examined PDCD4expression in endothelial cells under different type of shear stresses both in vivo and in vitro and performed gain and loss of function experiments to investigate the role of PDCD4in modulating turnover of vascular endothelial cells.ObjectivesWe investigated whether atheroprotective unidirectional pulsatile shear stress affects the expression of PDCD4in endothelial cells in vivo and in vitro and the role of PDCD4in modulating turnover of vascular endothelial cells.Materials and Methods MaterialsRabbit monoclonal antibody (mAb) for PDCD4, rabbit polyclonal antibody (pAb) for Cleaved Caspase-3and Caspase-3, rabbit mAb for p21Waf1/Cip1and mouse mAb for β-actin were from Cell Signaling Technology (Danvers, MA, USA). Rat mAb for PECAM-1were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse mAb for GAPDH and FITC-conjugated goat anti-rat IgG secondary antibodies were from ZSGB-BIO (Beijing). Alexa Fluor647-labeled goat anti-rabbit IgG secondary antibodies were from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). Rat tail collagen I was from BD Biosciences (San Jose, CA, USA). All other chemicals of reagent grade were from Invitrogen (Life Technologies, Carlsbad, CA, USA), unless otherwise noted.En Face Staining of Mice AortasFive C57BL/6mice (male,8-10weeks old,20-25g) were purchased from Peking University (Beijing). All animal experiments were performed in accordance with the Animal Management Rules of the Chinese Ministry of Health (document No55,2001) and with the approval of the Animal Care Committee of Shandong University.C57BL/6mice were deeply anesthetized with0.8%(wt/vol) pentobarbital sodium and transcardially perfused with30mL lukewarm saline, followed by100mL of4%paraformaldehyde. After perfusion, aortas were harvested and postfixed in this fixative solution for4hr and then subjected to en face immunostaining. Briefly, the adventitia was removed carefully, aortas were longitudinally dissected, blocked with5%(vol/vol) bovine serum albumin, then incubated with specific primary antibodies rat anti-CD31mAb and rabbit anti-PDCD4mAb at4℃overnight, then FITC-conjugated anti-rat IgG and Alexa Fluor647-conjugated anti-rabbit IgG secondary antibodies. Samples were counterstained with4’,6-diamidino-2-phenylindole (DAPI) for nuclei, mounted and photographed under a laser-scanning confocal microscope.Cell CultureHuman umbilical vein endothelial cells (HUVECs) were isolated from fresh human umbilical cords by trypsin perfusion. The cell pellet was resuspended in a culture medium consisting of medium199supplemented with20%(vol/vol) fetal bovine serum (FBS),2ng/ml fibroblast growth factor-2, and1%(vol/vol) penicillin/streptomycin. HUVECs (from passage4to7) were cultured in M199containing10%FBS with5%CO2at37℃for3days and then seeded onto glass slides precoated with collagen I.The study protocol conformed to the ethical guidelines of the1975Declaration of Helsinki with the approval of the Institutional Medical Ethics Committee of Qilu Hospital, Shandong University. All donors provided written informed consent.Flow ApparatusCells plated on collagen-coated slides were exposed to unidirectional pulsatile shear stress (1Hz) of12dynes/cm2or oscillatory shear stress (1Hz) of0±4dynes/cm2for0,1,3,6,9,12hr in a Streamer (Flexcell International Corp.), the parallel plate flow chamber, which was incorporated into a closed loop containing culture medium and kept in an incubator with5%CO2at37℃. A MasterFlex peristaltic pump (Cole Parmer, Vernon Hills, IL, USA) was applied to run the medium in the loop, and the type of shear stress was determined by the computer-controlled osci-flow apparatus (Flexcell International Corp.).Western Blot AnalysisCells were lysed with Cell lysis buffer for Western and IP and1mM phenylmethanesulfonyl fluoride. The total cell lysates were separated by SDS-PAGE and after incubation overnight at4℃with the designated antibodies, protein levels were analyzed by use of the LAS-4000luminescent image analyzer. Band densities were analyzed by use of Adobe Photoshop CS3.ImmunofluorescenceCells were fixed with4%paraformaldehyde and permeabilized with0.1%Triton X-100. After incubation overnight at4℃with anti-PDCD4antibodies, cells were incubated for1hr with Alexa647-conjugated secondary antibodies. Nuclei were stained with DAPI. Cells were examined under a laser-scanning confocal microscope.Transient TransfectionpEGFP-C1-Mock and pEGFP-C1-PDCD4plasmids were constructed and kindly provided by Dr. Olubunmi Afonja (New York University, New York). Transfection of vectors involved the Lipofectamine2000method.The sequence for PDCD4small interfering RNA (siRNA) was5’-GUGUUGGCAGUAUCCUUAG-3’. siRNA interference involved the Lipofectamine2000method. Bromodeoxyuridine (BrdU) Incorporation AssayProliferation was detected using the5-Bromo-2’-deoxy-uridine Labeling and Detection Kit I (Roche Diagnostics Corp, Indianapolis, IN, USA). Cells were treated with BrdU (10μM) for6hr before harvesting and were fixed at-20℃with Ethanol fixative, stained with Anti-BrdU working solution at4℃overnight, incubated with Anti-mouse-Ig-fluorescein or Alexa Fluor647-conjugated anti-mouse antibody for1hr at37℃and DAPI for8min at room temperature as a counterstain for the nucleus. The stained cells were examined using a laser-scanning confocal microscope. Proliferation was assessed based on the percentage of nuclei exhibiting BrdU incorporation.Apoptosis Detection (terminal deoxynucleotidyl transferase-mediated UTP nick end labeling, TUNEL)Apoptosis was detected using the ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, Billerica, MA, USA). Cells were fixed in1%paraformaldehyde and permeabilized in precooled ethanol:acetic acid2:1at-20℃. They were incubated with the Working Strength TdT Enzyme at37℃and then with the Anti-Digoxygenin Peroxidase Conjugate and Peroxidase Substrate to detect signs of apoptosis, staining brown. Counterstaining was carried out with methyl green. Apoptosis was assessed based on the percentage of TUNEL positive nuclei.Statistical AnalysisData are expressed as mean±SEM. Statistical analysis involved independent Student t test for comparing2groups and one-way ANOVA for multiple comparisons. P<0.05was considered statistically significant.ResultsPDCD4expression is maintained at a low level in areas of atheroprotective flow and induced in areas of atheroprone flow in vivoEndothelial cells were observed elongated and aligned with the longitudinal axis of the vessel in the straight segments of thoracic aortas, where shear stress is high and pulsatile; they showed a polygonal morphology in the inner curvature of aortic arches, where disturbed flow occurs with relatively low and oscillating shear stress or even under static conditions. Level of PDCD4was high in endothelial cell nuclei in the inner curvature of aortic arches, but was low in the straight segments of thoracic aortas. Thus, pulsatile shear stress in the native circulation maintains PDCD4expression at a low level in endothelial cells and oscillatory shear stress induces PDCD4expression in vivo.Pulsatile shear stress downregulates whereas oscillatory shear stress induces PDCD4protein level in HUVECsThe protein level of PDCD4was reduced with pulsatile shear stress since3hr (P<0.05) and induced with oscillatory shear stress since6hr (P<0.05) as compared with static control cells. Immunofluorescence of HUVECs revealed decreased PDCD4level in nuclei of cells under pulsatile shear stress (P<0.05) and induced under oscillatory shear stress (P<0.05) as compared with static conditions.PDCD4induces turnover (proliferation and apoptosis) of HUVECsOverexpression of PDCD4by transfecting cells with pEGFP-C1-PDCD4downregulated p21Waf1/Cip1protein level and increased proliferation as compared with mock treatment but upregulated that of Cleaved Caspase-3and induced apoptosis. Downregulation of PDCD4by siRNA did the opposite. Therefore, PDCD4induces turnover of HUVECs.Downregulation of PDCD4by pulsatile shear stress rescues p21Waf1/Cip1and reduces proliferation to slow the turnover of HUVECsHUVECs transfected with pEGFP-C1-PDCD4or pEGFP-C1-Mock were subjected to pulsatile shear stress or kept under static conditions. Pulsatile shear stress induced p21Waf1/Cip1protein expression and reduced proliferation. Overexpression of PDCD4blocked the induction of p21Waf1/Cip1and partly rescued proliferation reduction with pulsatile shear stress.HUVECs transfected with pEGFP-C1-PDCD4or pEGFP-C1-Mock were subjected to pulsatile shear stress or kept under static conditions. Pulsatile shear stress suppressed Cleaved Caspase-3and reduced apoptosis. Overexpression of PDCD4had no effect on the suppression of Cleaved Caspase-3or reduction of apoptosis with pulsatile shear stress.Upregulation of PDCD4by oscillatory shear stress reduces p21Waf1/Cip1and upregulates proliferation to maintain the turnover of HUVECsHUVECs transfected with PDCD4siRNA or negative control were subjected to oscillatory shear stress or kept under static conditions. Oscillatory shear stress reduced p21Wafl/Cipl protein expression and upregulated proliferation. Silense of PDCD4induced p21Waf1/Cipl and partly suppressed proliferation induction with oscillatory shear stress.HUVECs transfected with PDCD4siRNA or negative control were subjected to oscillatory shear stress or kept under static conditions. Oscillatory shear stress induced Cleaved Caspase-3and upregulated apoptosis. Silense of PDCD4had no effect on the induction of Cleaved Caspase-3or apoptosis with oscillatory shear stress.ConclusionsPulsatile shear stress reduces whereas oscillatory shear stress induces PDCD4protein expression in endothelial cells. PDCD4induces turnover (proliferation and apoptosis) of HUVECs. PDCD4level is associated with modulation of endothelial cell proliferation but not apoptosis under shear stresses. BackgroundVascular endothelial cells form a monolayer along the inner wall of blood vessels. Blood flows inside the tubes and generates shear stress against vessel wall surface. Shear stress activates receptors on the membrane of endothelial cells or in the extracellular matrix around and subsequently activates or suppresses specific signal pathways, modulates various gene expression and protein activity to keep the homeostasis of vascular endothelial cells. However, at bifurcations and curvatures of vessels or where the lumen is restricted, blood flow is disturbed and shear stress changes from laminar and pulsatile to low and oscillatory, which results in endothelial cell dysfunction and promotes onset of atherosclerotic plaques.Tumor suppressor programmed cell death4(PDCD4) was recently found important in cardiovascular cell biology, which inhibits proliferation and induces apoptosis of vascular smooth muscle cells, cardiac myocytes and fibroblasts, and represses contractile gene expression in vascular smooth muscle cells. Our above work showed that PDCD4promotes both proliferation and apoptosis of vascular endothelial cells. Chemical stimuli as well as mechanical stresses alter PDCD4expression. We have confirmed that shear stresses modulate protein level of PDCD4in vascular endothelial cells both in vivo and in vitro. However, the mechanisms involved remain unclear.Studies of tumor cells and several cardiovascular cells confirmed a negative correlation between microRNA-21(miR-21) and PDCD4. A conserved target site for miR-21was found within the3’untranslated region of PDCD4gene and miR-21blocked PDCD4expression by directly binding to the PDCD4mRNA. However, several other studies showed that refueling the starved cells with serum and tumor promoter12-O-tetradecanoylphorbol-13-acetate induced degradation of PDCD4protein via the ubiquitin-proteasome pathway. Both ways above suppress PDCD4expression post-transcriptionally. Little is known about modulation of PDCD4expression at the transcription stage. A putative binding site for nuclear factor kappaB (NF-κB) was found in the pdcd4gene in mice, but whether the sequence functions is unclear.The mechanism of PDCD4modulation by shear stresses is still under cover. Whether miR-21and the ubiquitin-proteasome system or other pathways are involved in the modulation remains to be clarified.ObjectivesWe investigated whether the reduction of PDCD4under pulsatile shear stress was via the ubiquitin-proteasome pathway and if NF-κB was involved in the induction of PDCD4under oscillatory shear stress.MethodsMaterialsRabbit monoclonal antibody (mAb) for PDCD4, Akt,(3-transducin repeat-containing protein (β-TrCP) and p65, rabbit polyclonal antibody (pAb) for ubiquitin, mouse mAb for phospho-Akt (Ser473),(3-actin and phosphatase and tensin homologue deleted on chromosome ten (PTEN), MG-132and LY294002were from Cell Signaling Technology (Danvers, MA, USA). Rabbit pAbs for70-kDa ribosomal protein S6kinase (p70-S6K) and phospho-p70-S6K (T412) were from Immunoway (Newark, NJ, USA). Protein A/G plus agarose were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit pAb for PDCD4(phospho S67) were from Abcam (Cambridge, UK). Mouse mAb for reduced glyceraldehyde-phosphate dehydrogenase (GAPDH) were from ZSGB-BIO (Beijing). Negative control microRNA inhibitor, miR-21inhibitor, negative control microRNA mimics and miR-21mimics were from GenePharma (Shanghai). Rat tail collagen I was from BD Biosciences (San Jose, CA, USA). Lactacystin was from Sigma (St. Louis, MO, USA). All other chemicals of reagent grade were from Invitrogen (Life Technologies, Carlsbad, CA, USA), unless otherwise noted.Cell CultureHuman umbilical vein endothelial cells (HUVECs) were isolated from fresh human umbilical cords by trypsin perfusion. The cell pellet was resuspended in a culture medium consisting of medium199supplemented with20%(vol/vol) fetal bovine serum (FBS),2ng/ml fibroblast growth factor-2, and1%(vol/vol) penicillin/streptomycin. HUVECs (from passage4to7) were cultured in M199containing10%FBS with5%CO2at37℃for3days and then seeded onto glass slides precoated with collagen I.The study protocol conformed to the ethical guidelines of the1975Declaration of Helsinki with the approval of the Institutional Medical Ethics Committee of Qilu Hospital, Shandong University. All donors provided written informed consent.Flow ApparatusCells plated on collagen-coated slides were exposed to unidirectional pulsatile shear stress (1Hz) of12dynes/cm2or oscillatory shear stress (1Hz) of0±4dynes/cm2for0,1,3,6,9,12hr in a Streamer (Flexcell International Corp.), the parallel plate flow chamber, which was incorporated into a closed loop containing culture medium and kept in an incubator with5%CO2at37℃. A MasterFlex peristaltic pump (Cole Parmer, Vernon Hills, IL, USA) was applied to run the medium in the loop, and the type of shear stress was determined by the computer-controlled osci-flow apparatus (Flexcell International Corp.). Western Blot AnalysisCells were lysed with Cell lysis buffer for Western and IP and1mM phenylmethanesulfonyl fluoride. The total cell lysates were separated by SDS-PAGE and after incubation overnight at4℃with the designated antibodies, protein levels were analyzed by use of the LAS-4000luminescent image analyzer. Band densities were analyzed by use of Adobe Photoshop CS3.RNA Isolation and Real-time PCRTotal RNA was extracted by use of TRIzol reagent. The first-strand cDNA was synthesized from2μg total RNA with use of random primers and the PrimeScript RT reagent kit (Takara Bio Inc.; Otsu, Shiga, Japan). Real-time PCR involved the SYRB Premix Ex Taq kit (Takara Bio Inc.). Primers for PDCD4were forward,5’-TGAGCACGGAGATACGAACGA-3’and reverse,5’-GCTAAGGACACTGCCAACACG-3’; and p-actin forward,5’-CGTGCGTGACATTAAGGAGA-3’and reverse,5’-CACCTTCACCGTTCCAGTTT-3’.β-actin was used as housekeeping gene. The relative mRNA expression level was assessed by the2-ΔΔCt method.ImmunoprecipitationCells were lysed with Cell lysis buffer for Western and IP and1mM phenylmethanesulfonyl fluoride. The same amount of protein from each sample was incubated with cognate antibodies for2hr at4℃, then with protein A/G plus agarose overnight at4℃with gentle rotation. The agarose-bound immunoprecipitates were rinsed and collected by centrifugation and incubated with the SDS-PAGE sample loading buffer (2×)(Beyotime Institute of Biotechnology), and subjected to SDS-PAGE and western blot analysis.Transient TransfectionThe sequence for the miR-21inhibitor was5’-UCAACAUCAGUCUGAUAAGCUA-3’and those for miR-21mimics were5’-UAGCUUAUCAGACUGAUGUUGA-3’and5’-AACAUCAGUCUGAUAAGCUAUU-3’. The sequence for β-TrCP siRNA were5’-GCACUUGCGUUUCAAUAAUTT-3’ and 5’-AUUAUUGAAACGCAAGUGCTT-3’; those for p65were5’-CGGAUUGAGGAGAAACGUATT-3’. MicroRNA inhibitor, mimics and siRNA interference involved the Lipofectamine2000method (Invitrogen).Statistical AnalysisData are expressed as mean±SEM. Statistical analysis involved independent Student t test for comparing2groups and one-way ANOVA for multiple comparisons. P<0.05was considered statistically significant.ResultsPulsatile shear stress post-transcriptionally reduces PDCD4expression in HUVECsWe examined both the protein and mRNA level of PDCD4and found that PDCD4protein level decreased gradually over time with pulsatile shear stress (P<0.05), however, with only a slight and non-significant increase in mRNA level (P<0.05). Therefore, pulsatile shear stress decreases PDCD4expression post-transcriptionally in HUVECs.Pulsatile shear stress reduces PDCD4protein level via the ubiquitin-proteasome pathwayThe proteasome inhibitor MG-132(10μM) or lactacystin (10μM) was added into the medium1hr before and during the shearing process with HUVECs, with dimethyl sulfoxide (DMSO) as a control. We also transfected the cells with β-TrCP siRNA. β-TrCP is an ubiquitin ligase involved in degradation of PDCD4. Either proteasome inhibitors or downregulation of β-TrCP completely rescued the PDCD4level downregulated with pulsatile shear stress (P<0.05), but neither affected PDCD4level under static conditions (P>0.05). Results of coimmunoprecipitation assays confirmed that pulsatile shear stress significantly induced ubiquitination of PDCD4as compared with oscillatory shear stress or static conditions (P<0.05). Hence, pulsatile shear stress reduces PDCD4protein level via the ubiquitin-proteasome pathway. We also transfected HUVECs with miR-21mimics or miR-21inhibitor (PTEN as a positive control, which is confirmed to be a target of miR-21in HUVECs), with no alteration of PDCD4expression (P>0.05), so the ubiquitin-proteasome pathway is more implied in the mechanism.Phosphatidyl inositol3-kinase (PI3K)/Akt pathway mediates the degradation of PDCD4by pulsatile shear stressHUVECs were pretreated with the specific PI3K inhibitor LY294002(10μM) or vehicle control for1hr, then were subjected to pulsatile shear stress for various times. Along with inhibition of Akt phosphorylation (P<0.05), activation of p70-S6K and phosphorylation of PDCD4were blocked (P<0.05), whereas PDCD4level was retained or even upregulated (P<0.05), which indicates that the PI3K/Akt pathway mediates the degradation of PDCD4by pulsatile shear stress.Oscillatory shear stress transcriptionally induces PDCD4expression in HUVECsWe examined both the protein and mRNA level of PDCD4and found that PDCD4protein level increased gradually over time with oscillatory shear stress, and PDCD4mRNA was also upregulated with a peak at6hr. Therefore, oscillatory shear stress increases PDCD4expression transcriptionally in HUVECs.Ubiquitin-proteasome-mediated degradation of PDCD4is not involved in the induction of PDCD4under oscillatory shear stressThe proteasome inhibitor MG-132(10μM) was added into the medium1hr before and during the shearing process with HUVECs, with DMSO as control. The proteasome inhibitor blocked but not promoted the PDCD4level induced by oscillatory shear stress (P<0.05), which indicates that oscillatory shear stress may not induce ubiquitin-proteasome degradation of PDCD4. Coimmunoprecipitation assays showed that oscillatory shear stress did not alter ubiquitination of PDCD4compared with static control (P>0.05). Phosphorylation of Akt was also induced but not suppressed by oscillatory shear stress (P<0.05) and no obvious difference was observed between Akt phosphorylation by oscillatory shear stress and that by pulsatile shear stress (P>0.05), which indicates that PI3K/Akt pathway is not involved in the induction of PDCD4by oscillatory shear stress. NF-κB is involved in the induction of PDCD4under oscillatory shear stressWe added NF-κB innibitor MG-132(10μM) into the medium1hr before and during the shearing process with HUVECs, with DMSO as control. We also transfected the cells with p65siRNA. We found that either NF-κB inhibitor or downregulation of p65blocked the PDCD4level induced by oscillatory shear stress (P<0.05), which indicates that NF-κB is involved in the induction of PDCD4under oscillatory shear stress.ConclusionsPulsatile shear stress induces ubiquitin-proteasome-mediated degradation of PDCD4via PI3K/Akt pathway in HUVECs. NF-κB is involved in the induction of PDCD4in HUVECs under oscillatory shear stress. |
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