Sestrin2 Restricts Endothelial-to-Mesenchymal Transition Induced by Lipolysaccharide via Autophagy

Objectives : Endothelial-to-mesenchymal transition (EndoMT) is a significant biological phenomenon wherein endothelial cells undergo a loss of their endothelial traits and progressively acquire mesenchymal characteristics. Consequently, this transformation leads to both a compromised ability to maintain lumen permeability and alterations in vascular structure, which hampers the preservation of blood-brain barrier integrity. This study aimed to investigate inflammation-induced EndoMT and its etiology, with the goal of impeding the infiltration of peripheral inflammation into the central nervous system. Materials and Methods : Lipolysaccharide (LPS) was administered intraperitoneally to mice several times to establish a chronic inflammatory model. A cellular inflammatory model was established by LPS in human brain microvascular endothelial cells (HBMECs). The mRNA expressions of inflammatory cytokines interleukin-1 β ( IL-1 β ) and IL-6 were detected by real-time polymerase chain reaction (PCR). Immunofluorescence staining of platelet endothelial cell adhesion molecule-1 (CD31) and alpha smooth muscle actin ( α -SMA) was conducted to assess the level of EndoMT. The expression levels of Occludin, zona occludens protein 1 (ZO-1), Sestrin2, microtubule-associated protein1 light chain 3 (LC3) and inducible nitric oxide synthase (iNOS) were detected by western blotting. Results : LPS treatment induced the downregulation of ZO-1 and Occludin, which was accompanied by the elevated expressions of iNOS, α -SMA, Sestrin2 and LC3-II in the mouse cortex and HBMECs. Mech-anistically, the knockdown of Sestrin2 in HBMECs exacerbated the EndoMT induced by LPS treatment, while the overexpression of Sestrin2 inhibited this process. Moreover, the induction of autophagy by rapamycin rescued the EndoMT induced by Sestrin2 knock-down. Conclusion : This study revealed that Sestrin2 inhibited endothelial inflammation and EndoMT via enhanced autophagy, which may provide a potential drug target for cerebrovascular inflammatory injury.


Introduction
The brain blood vessels serve as a vital interface for the exchange of nutrients, metabolites and gases between the peripheral and central nervous system (CNS), as well as a protective barrier against exogenous substances and neurotoxic components in blood [1].Vascular endothelial cells, which form a flattened epithelial layer along the internal surfaces of the heart, blood vessels, and lymphatics, exhibit phagocytic capabilities towards foreign bodies, bacteria, necrotic and senescent tissues, and actively participate in the immune response of an organism [2].When blood is contaminated with harmful substances, such as endotoxins and inflammatory factors, endothelial cells are initially subjected to abnormal blood environments, leading to impaired structure and function of these cells.Previous research has demonstrated that under the influence of various stimuli, endothelial cells undergo a transformation, losing their original characteristics and becoming deficient in tight junction proteins, thereby undergoing an endothelial-tomesenchymal transition (EndoMT).This transition is detri-mental to maintaining blood-brain barrier homeostasis and has the potential to facilitate the infiltration of peripheral inflammation into the CNS.Inhibition of EndoMT has significant alleviating effect on CNS disease [3][4][5].However, the majority of reports about EndoMT are focused on peripheral vascular and organ injuries, and EndoMT in the CNS requires further exploration.
Sestrins, a group of stress-induced proteins, exhibit a high degree of conservation across various organisms and are integral to cellular adaptation in response to stress [6].The diverse functions of Sestrins are observed in different organs and tissues.Sestrin2, a homologous protein to Sestrin1, is a remarkably conserved antioxidant protein initially recognized as hypoxia-inducing gene 95.Research has demonstrated that Sestrin2 accumulates in cells subjected to stress and assumes a crucial function in suppressing the generation of reactive oxygen species (ROS), consequently safeguarding cells against oxidative harm [7].As a stress-induced metabolic regulator, Ses-trin2 restrains oxidative stress and pro-inflammatory signaling pathways through mechanisms reliant on adenosine 5 ′ -monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) complex 1 [8,9].Previous researches have demonstrated the protective role of Sestrin2 in vascular-related diseases, wherein it activates AMPK and inhibits mTOR to preserve redox equilibrium [10,11].Inflammatory response, a characteristic feature of cardiovascular disease, is closely associated with oxidative stress, which results in endothelial dysfunction.Further research has shown that Sestrin2 alleviates oxidative stress and apoptosis of endothelial cells subjected to drug-induced endoplasmic reticulum stress [12].However, whether Ses-trin2 involves in EndoMT and its molecular mechanism are still uncovered.In this study, endothelial inflammation and EndoMT was induced by lipopolysaccharide (LPS) in vivo and in vitro.Here, it is shown that Sestrin2 inhibited endothelial inflammation and EndoMT via enhanced cell autophagy.Our results may provide a potential drug target for cerebrovascular inflammatory injury.

Animal Model
Male C57BL/6J mice (6-8-week-old) were obtained from the Experimental Animal Center of Nantong University.For the duration of the experiment, the mice were housed in a controlled environment that ensured pathogenfree conditions, consistent temperature and relative humidity.Treated mice were injected with 1 mg/kg/day LPS (L8880, Solarbio, Beijing, China) intraperitoneally for 5 days, while control mice were injected intraperitoneally with an equivalent volume of physiological saline for the same duration.The mice were subjected to anesthesia by isoflurane (R510-22-10, Rayward, Shenzhen, Guangdong, China) and underwent heart perfusion using physiological saline.Brain samples were gathered for further analysis.It is important to note that the animal experiments conducted in this study received approval and strictly adhered to the regulations established by the Animal Ethics Committee of Nantong University (S20230315-005).

Lentivirus Infection
Sestrin2 overexpressive lentivirus was constructed by GenePharma (Shanghai, China).HBMECs were plated in a 6-well plate at the confluence of 50% and infected by Ses-trin2 overexpressive lentivirus and control lentivirus with polybrene for 48 h.Then, HBMECs were exposed to LPS (100 ng/mL) for 24 h.

Transmission Electron Microscope
HBMECs were digested with 0.25% trypsin (C100C1, NCM Biotech, Suzhou, Jiangsu, China) containing ethylene diamine tetraacetic acid (EDTA) followed by centrifugation at 1200 rpm for 5 min.Cell pellet was resuspended with fixation fluid for electron microscope (G1102, Servicebio, Wuhan, Hubei, China) after discarding supernatant.Astrocytes were washed using sucrose-sodium cacodylate buffer and soaked in osmium tetroxide-sodium cacodylate for 2 h.Then, astrocytes were stained using 2% aqueous uranyl acetate after washing in water.Subsequently, samples were dehydrated in an ethanol series and embedded in Epon 812 (90529-77-4, SPI Science, West Chester, PA, USA).Ultrathin sections of 80 nm were obtained using a ultramicrotome with a diamond knife.Grids were poststained with 2% saturated uranyl acetate in 1% lead citrate and 50% ethanol.Photographs were captured using an electron microscope camera (TECNAI G2 20 TWIN, Thermo fisher, Waltham, MA, USA).

Statistical Analysis
The statistical analyses were conducted using Graph-Pad Prism 6.0 (GraphPad Software, San Diego, CA, USA).A Student's t-test was used to compare the difference between two groups and one-way analyses of variance (ANOVA) was used to compare the differences among multiple groups.The statistical difference was considered significant if p < 0.05.At least three replicates of each experiment were conducted, and all data were presented as mean ± standard deviation (SD).

Multiple Injections of LPS Induced Chronic Inflammation in Mouse Cortex
To investigate the mechanism of EndoMT in the inflammatory response, LPS was administered intraperitoneally to mice several times to establish a chronic inflammatory model.In comparison to the control group, the intraperitoneal injection of LPS significantly enhanced the expressions of iNOS and TNF-α in the mouse cortex (Fig. 1A).LPS treatment also induced the expressions of inflammatory factor IL-1β, IL-6 and IL-18 (Fig. 1B).Additionally, the mRNA expression levels of IL-1β (Fig. 1C) and IL-6 (Fig. 1D) were notably increased in the LPSinjected mice.

LPS Treatment Induced Endothelial Cell Dysfunction and Sestrin2 Expression in Mouse Cortex
After determining the inflammation induced by LPS, immunofluorescence staining of CD31 and α-SMA was performed to assess the level of EndoMT.As shown in Fig. 2A, a notable increase in the co-localization of CD31 and α-SMA was observed in the cortex of LPS-treated mice.In comparison to the control group, the expression levels of Occludin and ZO-1 were observed to decrease, while the expression level of α-SMA increased in the LPStreated group (Fig. 2B).Additionally, injection of LPS significantly enhanced the expression of Sestrin2 in the cortex of mice (Fig. 2C).These findings suggested that LPS treatment induced endothelial cell dysfunction and increased protein expression consistent with early EndoMT differentiation, accompanied by upregulated Sestrin2 expression.

Sestrin2 Overexpression Attenuated EndoMT in HBMECs
Subsequently, HBMECs were subjected to LPS exposure in vitro.This exposure led to an upregulation of iNOS and Sestrin2 expression in HBMECs, as depicted in Fig. 3A.To investigate the impact of Sestrin2 on En-doMT, specific Sestrin2 siRNAs were designed and transfected into HBMECs.Among these siRNAs, Sestrin2 siRNA 1 exhibited the most effective knockdown efficiency in terms of Sestrin2 expression, as shown in Supplementary Fig. 1A.Consequently, Sestrin2 siRNA 1 was employed in subsequent experiments.Notably, knockdown of  Sestrin2 exacerbated the reduced expression levels of Occludin and ZO-1 in the LPS-treated group (Fig. 3B).Simultaneously, knockdown of Sestrin2 facilitated the enhanced expression of α-SMA in the LPS-exposed group (Fig. 3B).Then, Senstrin2 overexpressive lentivirus was constructed and elevated the expression of Senstrin2 in HB-MECs (Supplementary Fig. 1B).Sestrin2 overexpression rescued the decreased expression levels of Occludin and ZO-1 in the LPS-treated group (Fig. 3C).Meanwhile, overexpression of Sestrin2 inhibited the enhanced expression of α-SMA in the LPS-treated group (Fig. 3C).These results suggested that Sestrin2 overexpression attenuated EndoMT in HBMECs.

Sestrin2 Regulated EndoMT Depending on Autophagy in Vitro
Subsequently, we aimed to investigate the downstream effects of Sestrin2 in the LPS-induced EndoMT process.As shown in Fig. 4A, the expression of microtubuleassociated protein1 LC3-II was observed to be elevated in the cortex of mice treated with LPS.Similarly, an increase in LC3B-II expression was observed in LPS-exposed HB-MECs (Fig. 4B).LPS treatment increased the autolysosomes (black arrows) in HBMECs (Fig. 4C).Overexpression of Sestrin2 facilitated the LC3-II expression in HB-MECs (Fig. 4D), whereas knockdown of Sestrin2 suppressed the expression of LC3-II in HBMECs (Fig. 4E).Furthermore, knockdown of Sestrin2 resulted in reduced expression levels of Occludin and ZO-1, which were initially inhibited by treatment with the autophagy inducer rapamycin (Fig. 4F).Additionally, the increased expression of α-SMA in the Sestrin2 siRNA-treated group was inhibited by rapamycin (Fig. 4F).Knockdown of Sestrin2 activated phosphorylation of mTOR, which was inhibited by rapamycin (Supplementary Fig. 2).These results suggested that Sestrin2 regulated EndoMT depending on mTOR-mediated autophagy.

Discussion
Vascular disease is thought to begin with endothelial dysfunction, which occurs at a preclinical stage, and is prone to complications [13].As a result of endothe-lial maladaptation to mechanical, metabolic, or oxidative stress, it causes impaired vasodilator response, altered angiogenesis, and increased expression of pro-inflammatory and pro-thrombotic factors [14].EndoMT is a cell phenotypic transition driven by inflammation and an important cytopathological manifestation of endothelial dysfunction, which plays an important role in the occurrence and development of chronic cardiovascular diseases [15].EndoMT is regulated by several signaling pathways, such as basic fibroblast growth factor/fibroblast growth factor receptor 1, transforming growth factor (TGF)-β, platelet derived growth factor receptor, and Wnt/β-Catenin signaling pathways [16].In this study, LPS significantly induced inflammatory reaction in mouse cortex and HBMECs, which was accompanied by notable EndoMT.This also suggests that an inflammatory reaction can induce EndoMT.Of course, endothelial cell dysfunction also exacerbates the progression of inflammation, and the two complement each other [17].
Oxidative stress is a prominent determinant of En-doMT, and its significance in the development of vascular pathogenesis has been extensively investigated.Suppression of EndoMT has been shown to diminish renal ROS levels and mitigate renal fibrosis [18].Sestrin2, a widely recognized antioxidant protein, assumes a critical function in curtailing the generation and buildup of ROS, thereby safeguarding cells against oxidative harm [19].Consequently, we hypothesized that Sestrin2 might be implicated in the induction of EndoMT through LPS treatment.In our study, it was revealed that Sestrin2 was upregulated in the presence of LPS in the mouse cortex and HBMECs.Sestrin2 siRNA was utilized in the rescue experiment conducted on HBMECs.Surprisingly, the knockdown of Sestrin2 did not suppress the EndoMT induced by LPS; rather, it facilitated its occurrence.Subsequent experiments corroborated that the overexpression of Sestrin2 safeguarded HBMECs against LPS-induced EndoMT.In summary, Sestrin2 exhibited a favorable impact on the preservation of endothelial cell function.The augmentation of Sestrin2 expression in response to LPS stimulation may represent an intrinsic protective mechanism initiated by the cells themselves.Nevertheless, the elevation of endogenous Sestrin2 expression was inadequate to manifest its protective influence.Other studies have found the similar result [20,21].LPS treatment significantly induced the upregulation of Sestrin2 in dendritic cells, and Sestrin2 overexpression suppressed the ferroptosis of dendritic cells in sepsis by inhibiting the activating transcription factor 4-CCAAT enhancer binding protein homologous protein signaling pathway [20].Similarly, Sestrin2 expression was induced in retinal ganglion cells exposed to H 2 O 2 , and upregulation of Sestrin2 significantly decreased H 2 O 2 -induced apoptosis and ROS generation [21].The protective role of Sestrin2 in different cells and tissues is hardly controversial.The differential expression of Sestrin2 in different disease models may be related to the degree of cell damage.
Autophagy is a self-degrading process that is crucial for balancing energy sources in response to nutritional stress and during vital periods of development [22].It serves as a crucial mechanism for cellular maintenance by eliminating misfolded or aggregated proteins, clearing damaged organelles such as mitochondria, endoplasmic reticulum, and peroxisome, and intracellular pathogens.Consequently, autophagy is commonly acknowledged as a survival mechanism, albeit excessive autophagy may also trigger cell death [23].Beyond its role in removing intracellular damaged or aggregated organelles, autophagy facilitates cell aging and cell surface antigen presentation, and prevents genomic instability and cell necrosis, thereby assuming a pivotal function in various disease processes.The available literatures indicate that autophagy plays a role in the regulation of EndoMT [3].However, the specific mechanism by which autophagy operates and the means by which it modulates EndoMT remain unclear.In human umbilical vein endothelial cells, the autophagy inhibitor 3-Methyladenine was found to suppress the EndoMT induced by SiO 2 and hypoxia [24].On the contrary, rapamycin counteracted the EndoMT process activated by TGF-β2 via reducing the phosphorylation level of Smad3 in human retinal microvascular endothelial cells [25], This finding strongly supports the conclusion that autophagy mitigates the EndoMT induced by LPS in HBMECs.As previously mentioned, there exist numerous pathways capable of activating EndoMT, and the induction of EndoMT in various cell models may involve distinct pathways.Furthermore, the roles of both moderate and excessive autophagy in cells differ significantly, which could contribute to the conflicting conclusion.
There is a limitation to our experiment as we were unable to definitively establish that the increased in Sestrin2 and inflammatory response in the mouse cortex induced by LPS were exclusive to endothelial cells.It is possible that these changes are a result of a mixed reaction involving multiple cell types in mouse brain.Subsequent cellular experiment confirmed the increased Sestrin2 and inflammatory response in HBMECs.In conclusion, the findings from both the animal and cell experiments provide support for our conclusion.

Conclusion
Our findings demonstrated that Sestrin2 effectively suppressed endothelial inflammation and EndoMT by promoting autophagy, thereby presenting a promising therapeutic target for cerebrovascular inflammatory injury.

Fig. 4 .
Fig. 4. Sestrin2 regulated EndoMT depending on autophagy in HBMECs.(A) Representative blots showed the LC3-II expression in the cortex of control and LPS-injected mouse.(B) Representative blots showed the expression of LC3-II in the control and LPSexposed HBMECs.(C) Transmission electron microscope photograph showed the autolysosomes (black arrows).Scale bar: 5 µm.(D) Representative blots showed the LC3-II expression in the control and LPS-exposed HBMECs with/without Sestrin2 overexpressive lentivirus infection.(E) Representative blots showed the LC3-II expression in the control and LPS-exposed HBMECs with/without Sestrin2 siRNA treatment.(F) Representative blots showed the LC3-II expression in the control and rapamycin-exposed HBMECs with/without Sestrin2 siRNA treatment.*p < 0.05 and **p < 0.01.Con, control; Rapa, rapamycin; LC3, light chain 3.