Impaired Autophagy Induced by oxLDL/β2GPI/anti-β2GPI Complex through PI3K/AKT/mTOR and eNOS Signaling Pathways Contributes to Endothelial Cell Dysfunction

Endothelial cell dysfunction plays a fundamental role in the pathogenesis of atherosclerosis (AS), and endothelial autophagy has protective effects on the development of AS. Our previous study had shown that oxidized low-density lipoprotein/β2-glycoprotein I/anti-β2-glycoprotein I antibody (oxLDL/β2GPI/anti-β2GPI) complex could promote the expressions of inflammatory cytokines and enhance the adhesion of leukocytes to endothelial cells. In the present study, we aimed to assess the effects of oxLDL/β2GPI/anti-β2GPI complex on endothelial autophagy and explore the associated potential mechanisms. Human umbilical vein endothelial cells (HUVECs) and mouse brain endothelial cell line (bEnd.3) were used as models of the vascular endothelial cells. Autophagy was evaluated by examining the expressions of autophagic proteins using western blotting analysis, autophagosome accumulation using transmission electron microscopy, and RFP-GFP-LC3 adenoviral transfection and autophagic flux using lysosome inhibitor chloroquine. The expressions of phospho-PI3K, phospho-AKT, phospho-mTOR, and phospho-eNOS were determined by western blotting analysis. 3-Methyladenine (3-MA) and rapamycin were used to determine the role of autophagy in oxLDL/β2GPI/anti-β2GPI complex-induced endothelial cell dysfunction. We showed that oxLDL/β2GPI/anti-β2GPI complex suppressed the autophagy, evidenced by an increase in p62 protein, a decrease in LC3-II and Beclin1, and a reduction of autophagosome generation in endothelial cells. Moreover, inhibition of autophagy was associated with PI3K/AKT/mTOR and eNOS signaling pathways. Rapamycin attenuated oxLDL/β2GPI/anti-β2GPI complex-induced endothelial inflammation, oxidative stress, and apoptosis, whereas 3-MA alone induced the endothelial injury. Our results suggested that oxLDL/β2GPI/anti-β2GPI complex inhibited endothelial autophagy via PI3K/AKT/mTOR and eNOS signaling pathways and further contributed to endothelial cell dysfunction. Collectively, our findings provided a novel mechanism for vascular endothelial injury in AS patients with an antiphospholipid syndrome (APS) background.


Introduction
As a complex and chronic progressive vessel disease, atherosclerosis (AS) is characterized by early endothelial dysfunction, and it leads to the morbidity of dysfunctional cardiovascular events worldwide [1][2][3]. Endothelial cell dysfunction plays a fundamental role in the pathogenesis of AS, resulting in the initiation of AS and the formation of ath-erosclerotic plaques [4]. The pathophysiological process of endothelial dysfunction is multifactorial and complex, and such process includes increased oxidative stress, abnormal autophagy, apoptosis, and inflammation [5][6][7][8].
Autophagy is an important biological process for lysosomal self-digestion, in which protein aggregates and damaged organelles are engulfed by double-membraned autophagosomes and transported to the lysosomes for degradation, thus, contributing to the maintenance of normal cellular function and survival [9][10][11]. Increased expressions of autophagy-degrading substrate p62, autophagy constituent protein LC3-II, and autophagy-related protein Beclin1 reflect the induction of autophagy [12]. The term autophagic flux represents the whole autophagic procedure, including autophagosome formation, transport of the autophagic substrate to lysosomes, and autophagosomal degradation in lysosomes [10]. The autophagic flux can be evaluated using inhibitors of autophagosome-lysosome fusion, such as chloroquine (CQ) and hydroxychloroquine (HCQ) [13]. Meanwhile, several molecular and cell signaling pathways have been implicated in regulating autophagy, such as PI3K/AKT/mTOR and AKT/eNOS pathways [14][15][16]. Under stressful conditions, such as starvation, hypoxia, and nutrient deficiency, the PI3K/AKT signaling pathway negatively regulates autophagy by mediating the mTOR expression in endothelial cells [17,18]. Moreover, eNOS is another classic downstream target of AKT, and the AKT/e-NOS signaling pathway is involved in the regulation of NO production and autophagy [16,19].
Accumulating evidence shows that proper autophagy has protective effects on the development of AS by participating in the regulation of cellular injury in endothelial cells and vascular smooth muscle cells (VSMCs) [20][21][22][23], although the underlying molecular mechanism remains largely unexplored. Previous experiments suggest that adequate endothelial autophagic flux limits the formation of atherosclerotic plaques by preventing endothelial apoptosis, senescence, and inflammation [5]. On the one hand, excessive activation of autophagy can result in endothelial cell death and plaque destabilization [24,25]. On the other hand, autophagy deficiency has been reported to increase the endothelial inflammation in patients with nonalcoholic steatohepatitis [26] and to accelerate the formation of atherosclerotic plaques in mice [22].
Antiphospholipid syndrome (APS) is an autoimmune disease characterized by thrombosis, pregnancy loss, and the presence of antiphospholipid antibodies (aPL), anti-β2 glycoprotein I antibodies (anti-β2GPI), and lupus anticoagulant (LA) [27,28]. β2GPI, which can bind to oxidized lowdensity lipoproteins (oxLDL) via domain V, is accepted as a potential autoantigen to accelerate the process of AS in patients with an APS background [29]. Based on these studies, it is hypothesized that oxLDL/β2GPI/anti-β2GPI complex, the combination of the oxLDL/β2GPI complex and anti-β2GPI, is a circulating immune complex that exerts a proatherogenic effect in patients with APS, which has been validated by published studies [30][31][32]. In previous studies, we demonstrated that oxLDL/β2GPI/anti-β2GPI complex could induce the foam cell formation of macrophages and VSMCs and the expressions of inflammatory cytokines in endothelial cells, resulting in the formation of atheromatous plaques with an APS background [33][34][35]. However, little evidence is available about the relationship between oxLDL/β2G-PI/anti-β2GPI complex and autophagy in endothelial cells. In the present study, we investigated the effects of autophagy on oxLDL/β2GPI/anti-β2GPI complex-induced endothelial dysfunction and its underlying molecular mechanisms.

Materials and Methods
2.1. Cell Culture and Treatment. HUVECs and bEnd.3 cells were purchased from the Shanghai Institutes for Biological Sciences. The cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Biological Industries, Israel) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Biological Industries, Israel), 4.5 g/L glucose, 1% glutamine, and 1% penicillin/streptomycin (Gibco, USA) at 37°C in a humidified atmosphere containing 5% CO 2 . All experiments were carried out when the cell density was 90-100%.
For the inhibition of the PI3K/AKT/mTOR pathway and eNOS pathway, the cells were pretreated with 10 μM PI3K inhibitor LY294002 (Sigma-Aldrich; USA), 1 μM AKT inhibitor AZD5363 (Abmole, USA), 1 μM mTOR inhibitor rapamycin (Abmole, USA), or 100 μM eNOS inhibitor L-NAME (Abmole, USA) for 4 h, followed by incubation in the presence or absence of oxLDL/β2GPI/anti-β2GPI complex for 24 h. Cells were pretreated with 10 μM CQ (Sigma-Aldrich, USA) for 4 h, followed by incubation in the presence or absence of oxLDL/β2GPI/anti-β2GPI complex for an additional 24 h. For the inhibition or activation of autophagy, the cells were treated with 5 mM 3-methyladenine (3-MA) (Abmole, USA) or 1 μM rapamycin in the presence or absence of oxLDL/β2GPI/anti-β2GPI complex for 24 h.  Table S1, and β-actin was selected as the housekeeping gene. The expressions of target genes were calculated using the 2 -ΔΔCt method.

Western
2.6. Enzyme-Linked Immunosorbent Assay (ELISA). HUVECs were seeded into 24-well plates at a density of 1:0 × 10 5 cells/well and treated with different stimuli as above described. The concentrations of IL-1β and IL-6 in the cell culture supernatants were analyzed using ELISA kits for IL-1β (Multiscience, China) and IL-6 (Multiscience, China) according to the manufacturers' instructions. The concentrations of the cytokines were expressed as pg/mL.

Intracellular Reactive Oxygen Species (ROS) Detection.
ROS was measured using the 2 ′ , 7 ′ -dichlorodihydrofluorescein diacetate (DCFH-DA) probe according to the manufacturer's instructions. Cells were incubated with 10 μM DCFH-DA (Jiancheng, Nanjing, China) at 37°C for 30 min. Cell images were captured by the BioTekCytation 5 Cell Imaging Multi-Mode Reader (BioTek, USA). Besides, the mean fluorescence of labeled cells was determined by flow cytometry (BD Biosciences, USA), with an excitation at 488 nm and emission at 530 nm. The data were analyzed using FlowJo software (version 10.0.7).
2.8. Intracellular Superoxide Dismutase (SOD) Detection. The activity of SOD in cells was examined using the xanthine oxidase method provided by the standard assay kit (Jiancheng, Nanjing, China). After stimulation, the total protein was collected using the supersonic schizolysis method, and the protein concentration was determined by a BCA Protein Assay Kit (Beyotime, Hangzhou, China). Samples were then determined according to the manufacturer's instructions. The values were expressed as units per mg protein, where one unit was defined as the amount of SOD inhibiting the reaction rate by 50% at 25°C.

Annexin V-FITC/PI Apoptosis Detection.
The apoptotic rate of HUVECs was detected using an AnnexinV-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit with propidium iodide (PI) (BD Biosciences, CA, USA) according to the manufacturer's instructions. After stimulation, the cells were digested with trypsin and suspended in 1× binding buffer at a density of 1 × 10 6 cells/mL. Cells were then stained with FITC annexin V and PI at room temperature for 15 min. Finally, samples were detected using flow cytometry (BD Biosciences, USA), and the total apoptotic rates (Q2 + Q3) were calculated. The data were analyzed using FlowJo software (version 10.0.7).
2.10. Statistical Analysis. All data were expressed as the mean ± SEM. Differences between the control and experimental conditions were assessed using the one-way ANOVA, followed by Tukey's multiple group comparison test. Twofactor treatment results were analyzed by two-way ANOVA with Tukey's test. Statistical analyses were performed with GraphPad Prism (version 7.0.0). Significant differences between the two groups were indicated by either an asterisk * or ns, where ns represents nonsignificance, * represents P < 0:05; * * represents P < 0:01; * * * represents P < 0:001, and * * * * represents P < 0:0001. All experiments were independently performed at least three times.

Effects
3.4. OxLDL/β2GPI/anti-β2GPI Complex Induces Autophagy Deficiency in PI3K/AKT/mTOR and eNOS Dependent Manner in Endothelial Cells. We investigated whether the signaling pathways explored above were involved in the effects of oxLDL/β2GPI/anti-β2GPI complex on suppressing autophagy of endothelial cells. We pretreated the cells with specific inhibitors of PI3K (LY294002), AKT (AZD5363), mTOR (rapamycin), or eNOS (L-NAME) and examined the effects of LY294002, AZD5363, rapamycin, or L-NAME on the expressions of autophagic proteins and the aggregation of LC3 puncta. We found that LY294002, AZD5363, rapamycin, and L-NAME pretreatment could significantly upregulate the expression of LC3-II, while downregulating the expression of p62 compared with the oxLDL/β2G-PI/anti-β2GPI complex alone group (P < 0 : 05; Figures 5(b) and 5(c)). Additionally, the aggregation of LC3 puncta in the oxLDL/β2GPI/anti-β2GPI complex group was notably increased after pretreatment with LY294002, AZD5363, rapamycin, and L-NAME (Figures 5(d) and 5(e)), indicating that inhibition of PI3K/AKT/mTOR and eNOS pathways reversed the suppressive effects of oxLDL/β2GPI/anti-β2GPI complex on the autophagy.

Activation of Endothelial Autophagy Decreases the
Expressions of oxLDL/β2GPI/anti-β2GPI Complex-Induced Inflammatory Cytokines in Endothelial Cells. Our previous study showed that oxLDL/β2GPI/anti-β2GPI complex is involved in the endothelial inflammatory response by promoting the expressions of various inflammatory cytokines   Oxidative Medicine and Cellular Longevity [35]. In the present study, we further investigated whether oxLDL/β2GPI/anti-β2GPI complex-induced .autophagy suppression was associated with endothelial inflammation by using autophagy activator rapamycin and autophagy inhibitor 3-MA. Consistent with previous studies [38,39], our results showed that the expression of LC3-II ( Figure S3(a)) and the aggregation of LC3 puncta ( Figure S3(b)) were increased after the treatment with rapamycin (1 μM), while it was decreased after the treatment with 3-MA (5 mM) in HUVECs. We found that rapamycin treatment downregulated the expressions of IL-1β, IL-6, and ICAM-1 at the mRNA level compared with the oxLDL/β2GPI/anti-β2GPI complex alone group (Figures 6(a)-6(c)). Meanwhile, 3-MA significantly increased the expressions of these inflammatory cytokines compared with the oxLDL/β2GPI/anti-β2GPI complex alone group (Figures 6(a)-6(c)). Moreover, the secretion of IL-1β and IL-6 and the expression of ICAM-1 followed a similar trend (Figures 6(d)-6(f)).

Activation of Endothelial Autophagy Prevents oxLDL/β2GPI/anti-β2GPI Complex-Induced Oxidative
Stress in Endothelial Cells. Oxidative stress due to ROS accumulation has been critically linked to endothelial dysfunction, leading to the progression of AS [40]. Therefore, we detected the generation of ROS using the ROS detection dye DCFH-DA. Fluorescence microscopy showed that the production of ROS was markedly increased in oxLDL/β2G-PI/anti-β2GPI complex-exposed cells, while the addition of rapamycin abolished the inductive effect of oxLDL/β2G-PI/anti-β2GPI complex on ROS production (Figure 7(a)).
The accumulation of ROS in the cells treated with autophagy inhibitor 3-MA was increased compared with the control group but not significantly changed compared with the oxLDL/β2GPI/anti-β2GPI complex alone treatment group (Figure 7(a)). We further confirmed these effects using flow cytometry and found that rapamycin could restore the promotion effects of oxLDL/β2GPI/anti-β2GPI compleximpaired autophagy on ROS production (Figures 7(c) and 7(d)). Meanwhile, the cells in the oxLDL/β2GPI/anti-β2GPI complex group and 3-MA group showed irregular shape with obscure borders, while the morphology of the cells was intact with a clear boundary after cotreatment with rapamycin and oxLDL/β2GPI/anti-β2GPI complex (Figure 7(b)). Moreover, we detected the generation of SOD. OxLDL/β2G-PI/anti-β2GPI complex significantly decreased the SOD activity compared with the control group, while the addition of rapamycin reversed the effect of oxLDL/β2GPI/anti-β2GPI complex on SOD activity (Figure 7(e)). Besides, the SOD activity in the 3-MA group was decreased compared with the control group, and it showed no significant difference compared with the oxLDL/β2GPI/anti-β2GPI complex alone group (Figure 7(e)).
Notably, the expressions of cleaved caspase-3 and cleaved caspase-9 at the protein level in the 3-MA group were further increased compared with cells treated with oxLDL/β2G-PI/anti-β2GPI complex alone (Figures 8(c)-8(e)), indicating that inhibition of autophagy could aggravate apoptosis.

Discussion
Previous studies have demonstrated that autoimmune response is involved in the pathogenesis of AS by contributing to the acceleration of atherosclerotic progression [42]. As an important immune complex in AS patients with an APS background, oxLDL/β2GPI/anti-β2GPI complex has been reported to have proatherogenic effects on most cells involved in AS, promoting the expressions of endothelial inflammatory cytokines, vascular smooth muscle cell prolif-eration, and macrophage foam cell formation [35,43,44].
With the discovery of autophagy, increasing attention has been paid to the alterations of autophagic flux during AS development and its relationship with AS-associated cellular damage [5,45,46]. Excess or deficiency in endothelial autophagy can result in cell death and injury and contribute to the formation of atherosclerotic plaques [5,47]. In the present study, we investigated how oxLDL/β2GPI/anti-β2GPI complex regulated autophagy and how the changes in autophagy modulated inflammation, oxidative stress, and apoptosis in endothelial cells. Western blotting analysis, TEM, and mRFP-GFP-LC3 tandem reporter assay were used to evaluate the expressions of autophagy-related proteins and autophagic processes in endothelial cells. It has been well established that Beclin-1, LC3, and p62 are the key proteins of autophagy, and Beclin1 and LC3II are upregulated during the activation of autophagy, accompanied by a decline of p62 [48]. Our study found  Figure 9: Proposed model for autophagy inhibition-promoted endothelial cell dysfunction induced by oxLDL/β2GPI/anti-β2GPI complex. OxLDL/β2GPI/anti-β2GPI complex, the circulating immune complex combined by oxLDL/β2GPI complex and anti-β2GPI, could suppress the autophagy process through activating both PI3K/AKT/mTOR and eNOS in endothelial cells. This inhibition would lead to elevated inflammation, oxidative stress, and apoptosis, which finally induced endothelial cell dysfunction. Abbreviations: oxLDL: oxidized lowdensity lipoprotein; β2GPI: β2-glycoprotein I; anti-β2GPI: anti-β2-glycoprotein I antibody; PI3K: phosphatidylinositol-3 kinase; AKT: serine/threonine kinase; mTOR: the kinase mammalian target of rapamycin; eNOS: endothelial nitric oxide synthase.  Table S1: genes and sequence of primer pairs used for RT-qPCR. Figure S1: effects of oxLDL/β2GPI/anti-β2GPI complex on the expressions of autophagy-related proteins at different time points in HUVECs. Figure S2: identification of the effects of AZD5363 on eNOS activation in HUVECs. Figure S3: identification of the effects of rapamycin and 3-MA on endothelial cell autophagy. (Supplementary Materials)