Low shear stress (LSS) is a well-established risk factor resulting in endothelial apoptosis and atherosclerosis. Autophagy has been reported to be involved in the development of atherosclerosis. However, whether autophagy participates in LSS-induced atherosclerosis remains unclear. The effect of autophagy and its association with apoptosis, in the development of atherosclerosis, remains controversial. Therefore, in the present study, the level and role of autophagy in human umbilical vein endothelial cells (HUVECs) exposed to LSS was examined. The results revealed that LSS increased the formation of autophagosomes and MAP1 light chain 3-like protein (LC3) puncta (as demonstrated by transmission electron microscopy and immunofluorescence), and the protein levels of Beclin-1 and LC3II decreased the expression of p62 [as revealed by western blot analysis (WB)]. Furthermore, the level of p62 decreased when autophagy was induced by rapamycin, and increased when autophagy was inhibited by chloroquine (CQ), which indicated that LSS may serve an important role in inducing autophagy flux. In addition, it was observed that HUVECs treated with LSS underwent apoptotic death, by monitoring the rate of apoptosis and the expression of apoptosis regulator BAX (Bax) and apoptosis regulator Bcl-2 (Bcl-2) (by flow cytometry and WB) and the LSS-induced apoptosis in HUVECs, that was significantly alleviated by pretreatment with rapamycin, partially via a decrease in the level of Bax and an increase in the level of Bcl-2. Pretreatment of HUVECs with CQ markedly increased LSS-induced apoptosis, which was associated with an increased expression of Bax and a decreased expression of Bcl-2. In conclusion, the results of the present study indicate that LSS increases the level of autophagy, which may be through a Bcl-2/Beclin-1-dependent mechanism, which serves a protective role against LSS-induced apoptosis.
It is well-known that atherosclerosis preferentially occurs at sites where the blood flow is slow or disturbed, and where the wall shear stress is low or oscillatory (
Autophagy is a highly regulated process, that may be involved in the turnover of long-lived proteins and organelles, and may help cells survive in an unfavorable environment (
Antibodies against MAP1 light chain 3-like protein (LC3; cat. no. L7543), rapamycin (cat. no. V900930), chloroquine (CQ; cat. no. C6628) and DAPI (cat. no. D9542) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Antibodies against apoptosis regulator Bcl-2 (Bcl-2; cat. no. sc7382), apoptosis regulator BAX (Bax; cat. no. sc70408), Beclin-1 (cat. no. sc48381) and β-actin (cat. no. sc47778) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antibody against p62 (cat. no. 5114 s) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). IR-Dye 680 (cat. no. 926-32220) or 800cw (cat. no. 926-32211) labeled secondary antibodies were purchased from Li-Cor Biosciences (Lincoln, NE, USA). The HUVECs were provided by the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). High-glucose Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Annexin-V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection kits were purchased from BD Biosciences (Franklin Lakes, NJ, USA).
The HUVECs were cultured in high-glucose DMEM supplemented with 10% FBS, in a 95% humidified incubator, with 5% CO2 at 37°C. For all of the experiments, HUVECs in passage 3 were used.
The flow experiments were performed as previously described (
At 70–80% confluency, the cells were treated with 5 nM rapamycin or 20 µM CQ for 24 h, followed by treatment with LSS (1.5 dyn/cm2) for an additional 0.5, 1, 2 or 3 h, respectively. The samples under static conditions (no flow) were used as the control.
Apoptosis in the HUVECs was measured with the Annexin V-FITC/PI Apoptosis Detection kit, according to the manufacturer's protocol. The stained cells were analyzed by flow cytometry (BD FACSAria III; BD Biosciences, Franklin Lakes, NJ, USA). Data analysis was performed using FlowJo version 7.6.1 (Tree Star, San Carlos, CA, USA).
Cells were seeded at a density 2×105 cells/well and fixed in 2.5% PBS glutaraldehyde at 4°C for 1 h. Post-fixation was performed in 1% OsO4, for 1 h. The cells were dehydrated in an ethanol gradient and embedded in Araldite (Huntsman Co., Ltd., Salt Lake City, UT, USA). Sections (40–60 nm) were placed on a grid (200 mesh) and were double-stained with uranylacetate and lead citrate. The sections were observed under a Philips CM-120 TEM.
The cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 5% non-fat milk for 2 h at room temperature, incubated with LC3antibodies (1:100) overnight at 4°C and stained with DAPI for 1 h, followed by incubation with FITC-conjugated secondary antibody (1:80, cat. no. ZF-0311, Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd., Beijing, China), immunoglobulin G, for 2 h. The images of the cells were captured using a fluorescence microscope (Leica TCS SP5). To quantify autophagic cells, LC3 puncta were determined in triplicate by counting >30 cells.
WB was performed as previously described (
All of the data were representative of at least three independent experiments and were expressed as the mean ± standard deviation. Statistical analyses were performed using one-way analysis of variance, followed by the Student-Newman-Keuls test. P<0.05 was considered to indicate a statistically significant difference. The statistical analysis was performed using SPSS software (version 18.0; SPSS, Inc., Chicago, Il, USA).
Increased LSS-only treatment times led to a gradual reduction in cell viability, with cell shrinkage and easy detachment from the coverslip compared with static cells (
Flow cytometry analysis demonstrated that LSS-only treatment resulted in a significant increase in apoptosis in a time-dependent manner (
Beclin-1, LC3 and p62 proteins are reliable markers for autophagy (
A normal cytoplasm, mitochondria and nuclei and a small number of autophagosomes and lysosomes were observed in the TEM images of the control group (
In addition, the treatment with LSS induced extensive formation of LC3 puncta compared with static cells, as determined by LC3 immunofluorescence staining (
In the present study, it was demonstrated that atheroprone LSS conditions were able to induce cell autophagy and apoptosis by regulating the balance of Bcl-2/Beclin-1 and Bcl-2/Bax. The induction of autophagy, by pretreatment with rapamycin, protected the HUVECs against LSS-induced apoptotic cell death. Autophagy inhibition by pretreatment with CQ resulted in elevated apoptotic cell death. With these results, it was concluded that autophagy served an important role in protecting against LSS-induced apoptosis.
Apoptosis is a highly regulated cell death process characterized by cell shrinkage, membrane blebbing, DNA fragmentation and chromatin condensation (
Autophagy, another type of programmed cell death, serves an important role in a number of physiological and pathological processes, including aging and cardiac ischemia (
Although Bcl-2 family proteins were initially characterized as cell death regulators, it has recently become clear that they also control autophagy. A study indicated that autophagy induction correlated with the dissociation of Bcelin-1 from Bcl-2 (
Although the upregulation of autophagy has been observed in HUVECs treated with LSS in the present study, it is unclear whether the autophagy is protective or detrimental. The cross talk between the autophagic and apoptotic cell death pathways is complex (
The results of the present study suggest that LSS was able to induce autophagy through the modulation of Bcl-2/Beclin-1 in HUVECs. Furthermore, it was observed that the cross talk between autophagy and apoptosis contributes to the autophagic protection of HUVECs from LSS-induced apoptosis. Although these results represent an advancement in the understanding of the association between LSS-induced autophagy and apoptosis, additional work is necessary to further characterize the protective effect of autophagy in the progression of atherosclerosis induced by LSS.
The authors would like to thank Dr Chunlai Wang (Harbin Veterinary Research Institute, Harbin, China) and Professor Zuyan Liu (Harbin Institute of Technology, Harbin, China) for their technical assistance. The present study was supported by the Natural Science Foundation of Heilongjiang Province Project (grant no. H201345) and by the Postdoctoral Foundation of Heilongjiang Province Project (grant no. LBH-Z11062).
Effects of LSS on the morphology of HUVECs. Cells were maintained under static conditions as controls or subjected to LSS (1.5 dyn/cm2) for (A) 0, (B) 0.5, (C) 1, (D) 2 or (E) 3 h. The cells were pretreated with RAPA (5 nM) (f-j) for 24 h and subjected to LSS (1.5 dyn/cm2) for (F) 0, (G) 0.5, (H) 1, (I) 2, and (J) 3 h. The cells were pretreated with CQ (20 µM) for 24 h and subjected to LSS (1.5 dyn/cm2) for (K) 0, (L) 0.5, (M) 1, (N) 2, and (O) 3 h. Images of the cellular morphology were captured using a microscope. Scale bar, 1,000 µm. HUVEC, human umbilical vein endothelial cells; LSS, low shear stress; RAPA, rapamycin; CQ, chloroquine.
LSS induces the apoptosis of HUVECs. (A) Results of apoptosis assay following treatment with (A-a) LSS, (A-b) RAPA and (A-c) CQ. (B) Quantification of the apoptosis data. The bar graphs represent the means ± standard error (n=3). *P<0.01 vs. the control. #P<0.01 vs. the cells pretreated with different modulators at the same point. LSS, low shear stress; HUVEC, human umbilical vein endothelial cells; RAPA, rapamycin; CQ, chloroquine; Con, control; PI, propidium iodide; FITC, fluorescein isothiocyanate.
LSS induces autophagy and apoptosis in HUVECs. The expression levels of the Beclin-1, LC3I, LC3II, p62, Bcl-2 and Bax proteins were determined by western blotting, following treatment with (A) LSS, (B) LSS+RAPA and (C) LSS+CQ. (D) Beclin-1 intensity, (E) LC3II/LC3I intensity, (F) p62 intensity, (G) Bcl-2 intensity and (H) Bax intensity were expressed as the fold change between LSS+RAPA, LSS+CQ and LSS. The bar graphs represent the mean ± standard error (n=3). *P<0.01 vs. the control. †P<0.05 and #P<0.01 vs. the cells pretreated with the various modulators at the same point. Con, control; LSS, low shear stress; HUVEC, human umbilical vein endothelial cells; RAPA, rapamycin; CQ, chloroquine; LC3, MAP1 light chain 3-like protein; Bcl-2, apoptosis regulator Bcl-2; Bax, apoptosis regulator BAX.
Ultrastructural changes induced by LSS in human umbilical vein endothelial cells. Cells were maintained under static conditions as controls at magnification (A) ×10,000 and (B) ×20,000, or subjected to LSS (1.5 dyn/cm2) for 1 h at magnification (C) ×10,000, (D) ×20,000 and (E) ×30,000. The square indicates the study area and is enlarged in the right panel to demonstrate the ultrastructure of the cells. The typical autophagosomes with the characteristic double membrane were shown. LSS, low shear stress; Con, control.
LSS induced autophagosome formation in HUVECs. (A) Cells were maintained under static conditions as controls or subjected to LSS (1.5 dyn/cm2) for (a) 0 h or (b) 1 h. The cells were pretreated with RAPA (5 nM) for 24 h and subjected to LSS (1.5 dyn/cm2) for (c) 0 h and (d) 1 h. The cells were pretreated with CQ (20 µM) for 24 h and subjected to LSS (1.5 dyn/cm2) for (e) 0 h and (f) 1 h. The LC3 puncta (autophagosomes) in the cells were visualized by fluorescence microscopy after immunofluorescence staining with an LC3 antibody, followed by a fluorescein isothiocyanate-labeled secondary antibody (green, original magnification ×400). The nucleus was stained with DAPI (blue, original magnification ×400). (B) Quantification of fluorescence intensity of LC3. Bar graphs represent average number of typical LC3 puncta/cell. Data are mean ± standard error from minimum 30 cells for each experiment (n=3). *P<0.01 vs. the control. #P<0.01 vs. the cells pretreated with the various modulators at the same point. LSS, low shear stress; HUVEC, human umbilical vein endothelial cells; Bax, apoptosis regulator BAX; LC3, MAP1 light chain 3-like protein; RAPA, rapamycin; CQ, chloroquine.