Selenoprotein S attenuates high glucose and/or ox-LDL-induced endothelium injury by regulating Akt/mTOR signaling and autophagy
Introduction
Diabetes mellitus (DM) is characterized by a continuously high blood sugar level; clinically, if unsuccessfully controlled, DM causes many complications, like cardiovascular disease, stroke, and chronic kidney, nerve, and eyes diseases, due to damages to endothelial cells (Cole and Florez, 2020, Faselis et al., 2020). To date, DM currently affects over 463 million people worldwide and the number of diabetics is expected to increase to 578 million by 2030 according to the latest International Diabetes Federation report (Huang et al., 2018). DM-induced vascular complications are a major cause for reduced people’s life expectancy and induced mortality (Zheng et al., 2018). Histologically, these vascular complications include damages to macrovessels, like atherosclerosis (AS) or endothelial dysfunction (ED) and the latter is a critically initial AS event and leads to defective endothelial repair and inability of angiogenesis (Xu et al., 2021). Diabetic patients usually complicate with dyslipidemia, the risk factors for AS (Poznyak et al., 2020). Previous studies revealed that high glucose and oxidized low-density lipoprotein (ox-LDL) could alter the oxidation-reduction equilibrium in vascular endothelial cells and induce them to undergo apoptosis; thus leading to ED (Lubrano and Balzan, 2014, Volpe et al., 2018). However, the underlying precise molecular mechanisms remain to be fully elucidated. In this regard, further identification and validation of novel approaches are urgently needed to effectively control DM and vascular complications in order to prevent or treat DM-related vascular complications.
DM-related endothelial damages could be due to increases in oxidation, inflammation, endoplasmic reticulum-related stress, apoptosis and autophagy disorders (Demirtas et al., 2016, Yuan et al., 2019). Autophagy is a tightly regulated metabolic process in cells and occurs in both physiologic and pathologic conditions and the latter includes diabetes (Demirtas et al., 2016). During autophagy, a cell digests cell own components via the lysosomal machinery, a survival-oriented function under base or stress conditions (Gross and Graef, 2020), which is resulted from a series of strictly regulated catabolic processes by which cytoplasmic components are transported to lysosomes and degraded and the digested cell components will be used to generate energy and protein synthesis (Cao et al., 2021). Cell components are tagged for degradation through ubiquitination and linked to microtubule associated protein 1 light chain 3 (LC3) through the adapter protein P62 (Wu and Lu, 2019). During autophagy initiation, LC3-I is lipidated to form LC3-II as an activated form of LC3 and then target the damaged cell components to form a double-membrane autophagosome (Hanada et al., 2007). Beclin-1 is a part of the lipid-phosphatidylinositol-3 kinase (PI3K) complex to regulate autophagosome formation and autophagy initiation (Kaur and Changotra, 2020). The contents of the autophagolysosome are degraded by cathepsins and P62 is degraded along with the targeted organelles and proteins (Cao et al., 2021, Kaur and Changotra, 2020). Thus, autophagy plays an important role in cell homeostasis and functions of heart and vessels, while defective or excessive autophagy could lead to diseases, like cardiovascular diseases or even carcinogenesis (Li et al., 2020, Mialet-Perez and Vindis, 2017). In DM a previous study reported that autophagy was able to protect high glucose- induced ED from senescence (Chen et al., 2014), while another previous report showed that autophagy played a detrimental role in hyperglycemia-induced ED (Niu et al., 2019). High glucose-induced autophagy could damage the endothelial progenitor cells, aggravate the mitochondrial oxidative stress, and prevent neovascularization (Kim et al., 2014). Moreover, ox-LDL was able to induce injury of human umbilical vein endothelial cells (HUVECs) via autophagy (Fan et al., 2015). Taken them all together, glycolipid metabolic disorders can cause endothelial injury via autophagy induction.
Selenoprotein S (SelS) is a transmembrane protein with extensive histological distributions, containing eight-serine and one-threonine phosphorylation sites (Gao et al., 2007). SelS is reported to be involved in regulation of the inflammatory response and oxidative and apoptosis during DM-induced macrovascular complications development and progression; therefore, SelS association with DM and macroangiopathy has gradually received attention from scholars in the field (Cui et al., 2018, Yu and Du, 2017, Yu et al., 2018, Zhong et al., 2020). Our group revealed that SelS upregulation was able to attenuate high glucose- and ox-LDL-induced endothelial injury in vitro (Zhong et al., 2020). However, it remains to be determined whether SelS endothelial protective effect is associated with autophagy and protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) signaling.
In this study, we assessed the role of SelS in alleviating glucolipid metabolic disorders-induced ED and changes in autophagy. We cultured human aortic endothelial cells (HAECs) in HG, HL and HGL and detected changes in HAECs damages, autophagy, and expression of the related proteins in vitro. We then manipulated SelS expression in HAECs and further cultured these HAECs in HG, HL, and HGL to further identify the role of SelS in HAECs in vitro. We expected to provide novel information regarding SelS in protection of endothelial cell damages induced by HG, HL, and HGL, which could create an experimental basis for SelS used as a targeted therapeutic strategy for protection of ED caused by glucolipid metabolism disorders in future.
Section snippets
Cells and culture
Primary HAECs were originally obtained from ScienCell (San Diego, CA, USA) and cultured in the endothelial cell medium (ECM; ScienCell) containing 5% fetal bovine serum (FBS) and endothelial cell growth supplements according to the manufacturer’s instructions in humidified incubator with 5% carbon dioxide (CO2) at 37 °C. The log-phased cells were further grown in various concentrations of HG, HL, and HGL-containing media.
Lentivirus and cell infection
Lentivirus carrying SelS cDNA, SelS short hairpin RNA (shRNA), or the
Establishment of an HAECs model of high glucose- and/or ox-LDL-induced injury
HAECs were grown in 25, 30, or 35 mmol/L glucose for 0, 3, 6, 12, 24, and 48 h. The cell viability was assayed using the MTT assay. As shown in Fig. 1A, HAECs viability was reduced after growth in 25, 30, and 35 mmol/L glucose for 48 h compared with the control group (Ctrl, glucose 5.5 mmol/L) and the mannitol control group (p < 0.05), especially in 35 mmol/L glucose (75.73% ± 3.58%). Our following experiments utilized 35 mmol/L glucose for 48 h as the optimally high glucose concentration and
Discussion
DM patients are frequently shown vascular complications due to DM-induced glucolipid metabolic disorders. In our previous 7-year prospective study of patients with primary type 2 DM (T2DM), we found that an intensive intervention for multiple factors could reduce the cumulative incidence of subclinical macroangiopathy by 69.09% (Yang et al., 2013) and DM-induced macrovascular complications could be due to multiple risk factors and the underlying pathological microenvironment for greater
Role of the funding source
The role of the funding sponsors does not involve any study designs, data collection,analysis, and interpretation or preparation of the manuscript, and manuscript submission for publication.
CRediT authorship contribution statement
ZW and YL performed experiments, analyzed data and wrote the paper; HY performed experiments; LM and JY wrote the paper; SY analyzed data; JD instructed the experiments and improved the manuscript. ZW and YL contributed equally to this work. All authors read and approved the final manuscript.
Declarations of interest
None.
Acknowledgements
This study was supported in part by grants from the National Natural Science Foundation of China (#82070831 and #81700726) and the Medicine Science Study Foundation of Dalian City (#2020QN019).
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