All cell lines were maintained at 37°C under humidified conditions and 5% CO₂. HUVECs were obtained from human umbilical cord veins as described (24). HUVECs were cultured as routine on gelatin-coated plastic dishes in M199 medium (Gibco, 31100-035) supplemented with 10% fetal bovine serum (FBS, Hyclone, SV30087.02) and 2 ng/mL fibroblast growth factor 2. All HUVECs involved in experiments were at no more than passage 10. HEK293T cells were cultured as routine in DMEM medium (Gibco, 12800–058) supplemented with 10% FBS.

Antibodies against c-Myc (sc-40), CD31 (sc-1506), α-actin (sc-32251), CD11C (sc-28671), CD206 (sc-58987), CD68 (sc-7084), normal mouse IgG (sc-2025), and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies of Grp94 (20292), LC3 (2775), PARP (9542), p-AMPK (2535), AMPK (5832), p-p70S6K (9205), p70S6K (9202), p-4EBP1 (9459), and 4EBP1 (9452) were from Cell Signaling Technology (USA). Antibody for His (66005-1) was purchased from Proteintech Group (USA) and the antibody of ACTB (122M4782) was from Sigma-Aldrich (USA). Secondary antibodies for immunofluorescence were goat anti-rat Alexa 488, goat anti-mouse Alexa 488, goat anti-rabbit Alexa 488, rabbit anti-goat Alexa 488, donkey anti-rabbit Alexa 546, and donkey anti-goat Alexa 546 were all from Invitrogen (USA).

HCP1 was synthesized as described (25) and dissolved in DMSO (10 mM, Sigma-Aldrich, D2650) as a stock solution. NLDL (BT-903) and oxLDL (BT-910) were purchased from Biomedical Technologies Inc. (USA). AICAR (S1802) was obtained from Selleck. cn (USA).

Cells were lysed in western and IP buffer (Beyotime, P0013) containing 150 mM NaCl, 20 mM Tris-HCl (PH 7.5), 1% Triton X-100, and proteinase inhibitors mix. After centrifuging at 4°C, the supernatant was collected. Protein samples (20 μg/lane) were loaded on 15% SDS-polyacrylamide gel and then transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, IPVH00010). The membranes were incubated with primary antibodies, then horseradish peroxidase-linked secondary antibodies and detected using an enhanced chemiluminescence detection kit (Thermo Fisher, 34080). The relative quantity of proteins was analyzed by Image J and normalized to loading controls.

Cells were fixed in 4% paraformaldehyde (w/v) for 15 min at room temperature or ice-cold methanol at −20°C for 10 min and blocked in phosphate-buffered saline (PBS), 0.01% Triton X-100 (v/v) and 5% donkey serum (v/v) for 60 min. Then cells were incubated with primary antibodies overnight at 4°C and washed in PBS three times followed by incubation with corresponding secondary antibodies for 1 h at 37°C. Fluorescence was detected by laser scanning confocal microscopy (Zeiss LSM700, Carl Zeiss Canada). ImageJ with WatershedCounting3D plug-in was used to obtain an objective number of LC3 puncta per cell. Tiff image was opened by ImageJ and the puncta count was recorded by WatershedCounting3D with parameters that allow optimal discrimination of signal/background (26). At least three images of each sample were quantitated and the experiment was repeated three times. The relative numbers of LC3 puncta per cell were normalized to the nLDL treatment group. The representative results are shown.

The coding region of Grp94 was sub-cloned into the pCMV3-C-Myc expression vector to produce the c-Myc-Grp94-wt construct. And the construct was confirmed by DNA sequencing. The His-tagged open reading frame clone of Homo sapiens AMPK (CH805185) was purchased from Vigene Biosciences (USA). Cells, at 70–80% confluence, were transfected with the expression vectors for 24 h by using Lipofectamine 2000 (Invitrogen, 11668–019) following the manufacturer's instructions.

Cells were plated and treated in 96-well plates followed by precipitating in 100 μl 10% trichloroacetic acid (Shenggong Biotech, Shanghai, China) for 1 h at 4°C. Then the cells were stained with 50 μl sulforhodamine B (SRB; Sigma-Aldrich, USA) for 10 min and the bounded dye was reconstituted in 100 μl of 10 mM Trisbase (pH 10.5). The optical density was read by a Spec-traMAX 190 microplate spectrophotometer (GMI Co., USA) at 540 nm. Cell viability (%) = (OD of treated group/OD of control group) × 100.

Cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in IP buffer (Beyotime, P0013) containing 150 mM NaCl, 20 mM Tris–HCl (pH 7.5), 1% Triton X-100, and proteinase inhibitors mix. After centrifuging at 4°C, the supernatant was collected and pre-cleared with protein A/G agarose beads (Beyotime, P2012) for 1 h at 4°C. Then the supernatant was gathered and incubated with specific primary antibodies or normal mouse IgG (as a control) and protein A/G beads overnight at 4°C. The beads were washed three times with IP buffer and then eluted with 2 × SDS loading buffer. And immunoprecipitated proteins were detected by western blot assay.

Cells were stained with Hoechst 33258 (Sigma-Aldrich, USA) for 10 min at 37°C. Then we washed them twice with PBS gently and photographed them with Olympus (Japan) BH-2 fluorescence microscope. At least three images of each sample were quantitated by image J and each experiment was repeated three times. The representative results are shown.

Male apoE^−/− mice (8 weeks old; C57BL/6J-knockout) used to build the atherosclerosis animal model were purchased from the Department of Laboratory Animal Science, Peking University Health Science Center (Beijing, China). The experimental design of this study was shown in Figure 5C. All procedures were under the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). The animal experimental protocol complied with the Animal Management Rules of the Chinese Ministry of Health (document no. 55, 2001) and was approved by the Animal Care Committee of Shandong University. Mice were fed with an atherogenic high-fat diet (21% fat and 0.15% cholesterol) for 20 weeks and randomized to three groups (n = 6 mice/group) for treatment. The baseline group was anesthetized by isoflurane inhalation (3%) plus 1 L/min O₂ and euthanized by exsanguination/cervical dislocation wherever appropriate. The HCP1 treatment group received 8 weeks' intraperitoneal injections of HCP1 (1 mg/kg per day). The Control group was injected with the same volume of dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA) diluted with PBS.

The bodyweight of mice was measured once a week during HCP1 treatment. For mice at 28 and 36 weeks of age, animals were anesthetized by isoflurane inhalation (3%) plus 1 L/min O₂ and euthanized by exsanguination/cervical dislocation wherever appropriate. The aorta and heart were rapidly extracted. The heart including aortic roots was embedded in optimal cutting temperature (OCT) embedding medium (Tissue-Tek, Torrance, CA, USA) for following histology and immunofluorescence assay. The adventitia was thoroughly stripped, and the remaining aorta was opened longitudinally and fixed in 4% paraformaldehyde for measurements of plaque surface area. Other organs including the heart, liver, spleen, lung, kidney, brain, pancreas, and thymus were removed and weighed. The organ coefficient was obtained by dividing the weight of the organ by body weight.

The aortic roots of mice were embedded in an OCT embedding medium and cryosections of the aortic sinus (7 μm) were prepared. Aortic root cryosections underwent Masson's trichrome staining (Sigma-Aldrich). Images of cryosections were taken with a digital camera and analyzed by ImagePro Plus. The corresponding sections were stained with primary antibodies (1:100) overnight at 4°C and incubated with the appropriate secondary antibodies (1:200) for 1 h at 37°C. Then samples were observed by laser scanning confocal microscopy (Zeiss LSM700, Carl Zeiss Canada). Six samples were detected in each group and at least three images of each sample were quantitated. Carl Zeiss AxioVision 4.6 was used to measure the fluorescence intensity of each aortic root sample. The representative results are shown.

Cryosections (7 μm) of mouse aortic roots were incubated with 10 μg/ml quenched FITC-labeled DQ gelatin (Invitrogen) and 1 μg/ml propidium iodide (PI, Sigma-Aldrich) in 0.5% low melting point agarose (Invitrogen), cover-slipped, and chilled for 5 min at 4°C. Then sections were incubated at 37°C for 2 h and observed by fluorescence microscopy. Gelatinase inhibitor (MMP-2/9 inhibitor IV, Chemicon, Millipore) was added as a control. Then samples were observed by laser scanning confocal microscopy (Zeiss LSM700, Carl Zeiss Canada). Six samples were detected in each group and at least three images of each sample were quantitated. Carl Zeiss AxioVision 4.6 was used to measure the fluorescence intensity of each aortic root sample. The representative results are shown.

The fixed ascending aorta and proximal arch segment were incubated with 10% donkey serum for 30 min. Each aortic arch was then incubated simultaneously with rabbit-anti-LC3 and goat-anti-CD31 overnight at 4°C and incubated with the appropriate secondary antibodies: rabbit anti-goat Alexa 488 and donkey anti-rabbit Alexa 546 for 1 h at 37°C. The nuclei were stained with DAPI. Then aorta segments were pinned flat with the endothelium facing up on glass slides, and LC3 patches were detected by laser scanning confocal microscopy (Zeiss LSM700, Carl Zeiss Canada). At least three images of each sample were quantitated by image J and six samples were detected in each group. The representative results are shown.

Apoptotic cells in the atherosclerotic lesion were detected by TUNEL using an in situ cell death detection kit (Sigma, 12156792910) according to the manufacturer's instructions and sections were counterstained with DAPI to detect nuclei. Then samples were detected by laser scanning confocal microscopy (Zeiss LSM700, Carl Zeiss Canada). At least three images of each sample were quantitated by image J and six samples were detected in each group. The representative results are shown.

Results are reported as mean ± SEM. We used sample sizes of 6 mice per group (α = 0.05). All experiments were repeated at least three times independently. For statistical analysis, Graph Pad Prism software (version 5.0) was used. Statistical comparisons for data were performed using student's t-test and one-way analysis of variance (ANOVA) followed by Tukey: compare all pairs of columns test. Differences were regarded as statistically significant when p < 0.05.

Firstly, we detected the effect of HCP1 on oxLDL-induced VEC injury. Western blot analyses showed that oxLDL increased Grp94 protein level in human umbilical vein endothelial cells (HUVECs) (Figure 1A). Treatment with HCP1 inhibited cell viability reduction induced by oxLDL in HUVECs (Figure 1B). These results suggested that HCP1 suppressed oxLDL-induced HUVEC injury.

Then we examined the effect of HCP1 on oxLDL-induced autophagy in HUVECs. Microtubule-associated protein 1 light chain 3 (MAP1LC3/LC3) is a ubiquitin-like protein involved in the formation of autophagosomes. The PE-conjugated form LC3-II is the only protein marker that is reliably associated with completed autophagosomes as well as phagophores. Therefore, LC3 is referred to as an autophagosome marker (27). Treatment with HCP1 (1 μM, 2 μM) decreased the number of LC3 dots per cell (Figure 2A) as well as the protein level of LC3-II (Figure 2B). Furthermore, transfection with c-Myc-Grp94-wt plasmid blocked the effect of HCP1 compared with the empty vector-transfected groups (Figure 2C). These results suggested that inhibition of Grp94 by HCP1 suppressed oxLDL-induced autophagy in HUVECs.

OxLDL induced ROS generation and autophagy in HUVECs and the autophagic response was reported to be mediated by the ROS-LOX-1 pathway (4). ROS could activate adenosine monophosphate-activated protein kinase (AMPK) through two key upstream kinases calcium-dependent protein kinase 2 or serine threonine kinase 11 (28, 29). In addition, HSP90 was found to interact with AMPK and maintain its AMP-activated kinase activity (30).

Therefore, we detected whether Grp94 could interact with AMPK and influence its activity. The interaction between Grp94 and AMPK was detected in HEK293T cells and HUVECs by co-immunoprecipitation experiments. Results showed that Grp94 might interact with AMPK and treatment with HCP1 did not influence the interaction between them (Figures 3A,B). AMPK is activated by the phosphorylation of Thr172 in the α subunit (31). Treatment with HCP1 inhibited AMPK activity by decreasing the phosphorylation of AMPK (Thr172) in HUVECs (Figure 3C). Furthermore, transfection with c-Myc-Grp94-wt plasmid blocked the effect of HCP1 on AMPK activity compared with the empty vector-transfected groups (Figure 3D). These results suggested that inhibition of Grp94 by HCP1 suppressed AMPK activity in HUVECs.

MTORC1 is a master negative regulator of autophagy and AMPK inhibits mTORC1 activity by phosphorylating TSC2 and RAPTOR (32). In accordance with previous studies, we found that treatment with oxLDL inhibited mTORC1 activity and reduced the phosphorylation of mTORC1 downstream targets ribosomal protein S6 kinase (p70S6K) and 4E-binding protein 1 (4EBP1) (11). Therefore, we detected the effects of HCP1 on mTORC1 activity. Treatment with HCP1 elevated the phosphorylation of p70S6K and 4EBP1 indicated that HCP1 activated mTORC1 activity (Figure 4A). Furthermore, co-treatment with AMPK activator acadesine (AICAR) eliminated the effect of HCP1 on mTORC1 activity suggested that HCP1 inhibited the AMPK-mTORC1 pathway. In addition, AICAR also inhibited the effect of HCP1 on protein levels of LC3-II (Figure 4B). These results demonstrated that HCP1 inhibited oxLDL-induced autophagy dependent on the AMPK-mTORC1 pathway.

Excessive activation of autophagy can lead to apoptosis (33). The effects of HCP1 on oxLDL-induced apoptosis were determined. Poly (ADP-ribose) Polymerase (PARP) is a family of 17 proteins and PARP1 is the most extensively studied one. PARP1 participates in several cell stress processes, especially DNA damage repair. It can bind to DNA signal- and double-strand breaks, then parylate histones and other DNA repair proteins as part of the DNA repair mechanism. During apoptosis, caspases cleave PARP1 and separate the catalytic domain from the DNA binding domain. PARP1 becomes inactive and loses its ability to respond to DNA damage, resulting in cell death (34). HCP1 reduced protein levels of cleaved PARP (Figure 5A) and apoptosis rate (Figure 5B) in HUVECs indicated that HCP1 suppressed oxLDL-induced apoptosis.

Endothelial dysfunction is closely associated with atherosclerosis. To further elucidate the role of Grp94 in atherosclerosis, we examined the effect of HCP1 on the atherosclerotic plaque in apoE^−/− mice (Figure 5C). Firstly, we assessed the level of autophagy in the endothelium of advanced atherosclerotic lesions by en face immunofluorescent staining of LC3 dots. Results showed that the number of LC3 puncta per cell was significantly decreased in the HCP1 treatment group (Figure 6A), suggesting that HCP1 inhibited autophagy of atherosclerotic vascular endothelium in apoE^−/− mice. In addition, we detected and quantified apoptosis in the endothelium of plaque by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. Results showed that treatment with HCP1 inhibited vascular endothelium apoptosis in plaque (Figure 6B). Therefore, HCP1 suppressed autophagy and apoptosis in the endothelium of apoE^−/− mice.

Unstable plaques are typically presented with a large necrotic core covered by a thin fibrous cap and a highly inflammatory cell content (21). Endothelial cell, macrophage, and smooth muscle cell apoptosis in plaque contributes to the expansion of necrotic core. Treatment with HCP1 inhibited cell apoptosis of the whole plaque including endothelial cells, macrophages, and smooth muscle cells (Supplementary Figure 1). Smooth muscle cells are predominant in the fibrous cap and thicken atherosclerotic lesions by producing collagen, elastin, and other matrix components. Apoptosis of smooth muscle cells in the fibrous cap reduces extracellular matrix protein production and leads to thinner fibrous caps. Compared with the control group, mice treated with HCP1 showed increasing plaque collagen content and smooth muscle cells (Figures 7A,B). The proteolytic enzymes matrix metalloproteinases play important roles in weakening the fibrous cap and promoting plaque rupture. HCP1 treatment reduced the activity of MMP-2/9 of the atherosclerotic lesion in apoE^−/− mice (Figure 7C). Smooth muscle cell apoptosis also exacerbates plaque inflammation and the inflammatory macrophages can induce smooth muscle cell apoptosis. Treatment with HCP1 decreased pro-inflammatory macrophages known as M1 while increased anti-inflammatory macrophages known as M2 (Figure 7D) (35). Therefore, HCP1 promoted the stabilization of atherosclerotic lesion.

To determine whether there is potential toxicity of HCP1 to apoE^−/− mice, toxicity experiments were carried out. We evaluated the influence of HCP1 on body weight and organ coefficients including heart, liver, spleen, lung, kidney, brain, pancreas, and thymus. The results showed that there were no significant differences in body weight and organ coefficients between the control group and HCP1-treated group (Supplementary Figure 2).