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Hydrogen sulfide (H2S), mainly produced by cystathionine-gamma-lyase (CSE) in vascular system, emerges as a novel gasotransmitter and exerts anti-atherosclerotic effects. Alterations of CSE/H2S pathway may thus be implicated in atherogenesis. However, the cellular and molecular mechanisms remain to be clarified. In this study, we showed that ox-LDL, the major risk factor of atherosclerosis, suppressed CSE promoter activity, and decreased its mRNA and protein expression, as well as H2S generation in macrophage. Exogenous H2S and CSE overexpression reduced TNF-alpha and ICAM-1 generation in ox-LDL-treated macrophage and its adhesion to the endothelial monolayer, whilst CSE knockdown produced opposite effects. Interestingly, ox-LDL increased the expression and activity of DNA methyltransferase (DNMT) and resulted in hypermethylation of CpG islands in the cse gene promoter region, as determined by mehylation-specific PCR and bisulfate sequencing. Moreover, DNMT inhibitor 5-aza-2′-deoxycytidine and DNMT1 siRNA abolished the CSE decrease induced by ox-LDL. More important, the ox-LDL-induced disturbance of CSE and DNMT was confirmed in the peritoneal macrophage isolated from ApoE−/− mice. These results reveal that DNA hypermethylation may be involved in ox-LDL induced CSE downregulation and subsequent H2S reduction in macrophage. We conclude that ox-LDL activates macrophage and triggers vascular inflammation by suppressing CSE transcription via its promoter region hypermethylation. [ABSTRACT FROM AUTHOR]

Objective: Our previous studies found that 100 µg/ml oxidized low-density lipoprotein (ox-LDL) could up-regulate the autophagic level in human umbilical vein endothelial cells (HUVEC). The present study was conducted to observe the roles of oxidative stress and lectin-like oxidized low density lipoprotein-1 (LOX-1) in the ox-LDL-induced up-regulation of autophagy., Methods: Prior to the ox-LDL exposure, LOX-1mAb, vitamin C and vitamin E were used to study the roles of LOX-1 and oxidative stress in the activation of autophagy. The contents of total-superoxide dismutase (T-SOD) and MDA (malondialdehyde) in the culture medium were detected with enzyme linked immunosorbent assay. Western blot was employed to detect the levels of autophagic marker microtubule-associated protein light chain 3 (MAP1-LC3)-II/LC3-I, beclin1 and lysosome associated membrane protein 2a (lamp2a)., Results: After the ox-LDL exposure, the down-regulated level of T-SOD [0.5 h (32.73 ± 1.09 vs 40.16 ± 1.28) U/ml, P < 0.01; 6 h (29.32 ± 1.56 vs 40.16 ± 1.28) U/ml, P < 0.01] and the up-regulated level of MDA [0.5 h (1.11 ± 0.04 vs 0.57 ± 0.05) nmol/ml, P < 0.01; 6 h (0.69 ± 0.03 vs 0.57 ± 0.05) nmol/ml, P < 0.05] in culture medium were also significant at 0.5 h and 6 h. The ox-LDL-induced increased ratio of LC3-II/LC3-I was reversed by the pretreatments of vitamin C and vitamin E (0.5 h, vitC: 3.11 ± 0.02 vs 4.31 ± 0.50, P < 0.05; vitE: 3.46 ± 0.19 vs 4.31 ± 0.50, P < 0.05; 6 h, vitC: 1.44 ± 0.05 vs 2.31 ± 0.16, P < 0.05), but not LOX-1mAb. LOX-1mAb decreased the ox-LDL-induced elevated level of lamp2a protein while vitamin C and vitamin E only inhibited the elevation of lamp2a at the timepoint of 6 h, but not 0.5 h., Conclusion: Oxidative stress, rather than LOX-1, plays an important role in the ox-LDL-induced up-regulation of autophagy in HUVEC. The formation of autolysosomes is associated with the LOX-1-mediated endocytosis of ox-LDL. Oxidative stress only plays a minor role in the formation of autolysosomes induced by the engulfed ox-LDL.

Advanced glycation end-products (AGEs) contribute to the pathogenesis of diabetes mellitus and atherosclerosis by promoting vascular endothelial cell proliferation, migration, damage and death. In this study, we examined the role of autophagy in HUVECs exposed to AGE-modified bovine serum albumin (AGE-BSA). HUVECs incubated with AGE-BSA for 6 h showed an increase in the formation of acidic vesicular organelles and autophagosomes. AGE-BSA-induced upregulation of microtubule associated protein 1 light chain 3-II (LC3-II), a marker of autophagy, was abolished by pretreatment with the autophagy inhibitor 3-methyladenine (3-MA), and was increased by rapamycin, an autophagy inducer. The increase of lactate dehydrogenase (LDH) leakage induced by AGE-BSA was increased by 3-MA, but not rapamycin. An oxidative inhibitor, α-tocopherol, decreased not only the AGE-BSA-induced increase of reactive oxygen species, but also the upregulation of LC3-II protein levels. These results suggest that AGE-BSA increases the level of autophagy, which is protective against HUVEC injury, and that ROS play a role in this activation of autophagy.

Objective: To investigate the role of autophagy in oxidized low density lipoprotein (ox-LDL) induced injury of human umbilical vein endothelial cells (HUVEC)., Methods: The cultured HUVECs were randomly divided into four groups of control, ox-LDL, ox-LDL + rapamycin and ox + 3-methyladenine (3-MA). The cells were used to detect the ratio of LC3-II/LC3-I by Western blot while the proliferation and apoptosis of cells measured by MTT and flow cytometry. The lactate dehydrogenase (LDH) activity and endothelin-1 (ET-1) content in the supernatant were detected with enzyme linked immunosorbent assay., Results: The Ox-LDL treatment up-regulated the ratio of LC3-II/LC3-I in HUVEC (P < 0.01). It increased the activity of LDH (P < 0.01)and content of ET-1 (P < 0.05) in the supernatant. Also it induced the proliferation (P = 0.028) and apoptosis (P < 0.05) of cells. The autophagic inducer rapamycin increased the up-regulation of autophagic level induced by ox-LDL, decreased the activity of LDH and content of ET-1 (P < 0.05) and inhibited the ox-LDL-induced proliferation of cells. Conversely, the autophagic inhibitor 3-MA decreased the elevation of LC3-II/LC3-I induced by ox-LDL (P < 0.01) and increased the cell apoptosis and death., Conclusion: Ox-LDL exposure can increase the secretion of LDH and ET-1 and induce the proliferation and apoptosis of cells so as to cause a harmful effect in the survival of HUVEC. The injury may be reduced by rapamycin, an autophagic inducer, and elevated by the autophagic inhibitor 3-MA.