Melatonin inhibits vascular endothelial cell pyroptosis by improving mitochondrial function via up-regulation and demethylation of UQCRC1.

Atherosclerosis (AS) is a chronic inflammatory disease that involves cell death and endothelial dysfunction. Melatonin is an endocrine hormone with anti-inflammatory and anti-AS effects. However, the underlying molecular mechanisms for the anti-AS effects of melatonin are unknown. A previous study has shown that pyroptosis plays a detrimental role in the development of AS, therefore, this study was designed to investigate the anti-pyroptotic effects and potential mechanisms of melatonin in atherosclerotic endothelium. Our results show that melatonin attenuated the expression of genes related to pyroptosis, including NLRP3, caspase-1, and IL-1ß, in human umbilical vein endothelial cells treated with oxidized low-density lipoprotein. Furthermore, melatonin up-regulated the expression of ten-eleven translocation 2 (TET2), inhibited the methylation of ubiquinol-cytochrome c reductase core protein 1 (UQCRC1), and reduced pyroptosis. The up-regulation of UQCRC1 by melatonin improved mitochondrial function, thereby inhibiting oxidative stress and endothelial cell pyroptosis. Collectively, our results indicate that melatonin prevents endothelial cell pyroptosis by up-regulating TET2 to inhibit the methylation of UQCRC1 and improving mitochondrial function.

High glucose condition inhibits trophoblast proliferation, migration and invasion by downregulating placental growth factor expression.

AIM: This study aimed to investigate the effect of high glucose (HG) level on the proliferation, migration and invasion of trophoblasts and determine the role of placental growth factor (PLGF) in the process. METHODS: HTR8-S/Vneo was treated with different concentrations of d-glucose (0, 10, 15, 20, 25 and 30 µM) at different times (0, 6, 12 and 24 h). qRT-PCR and Western blot analyses were used to measure PLGF expression. The protein level of PLGF was measured by immunofluorescence. Cell proliferation was assessed with CCK-8 analysis. Wound healing and transwell assays were used to evaluate cell migration and invasion. Intercellular ROS was detected with DCFH-DA. RESULTS: After d-glucose treatment, the viability decreased in 25 and 30 µM groups. The HG group (25 µM) showed inhibited cell migration and invasion ability. The mRNA and protein levels of PLGF decreased under HG condition. Elevated ROS production was also detected in the HG group. Knocked-down PLGF expression enhanced increased ROS production and decreased cell migration and invasion, which reverted to the original levels after PLGF was overexpressed. CONCLUSION: High glucose treatment inhibited HTR8-S/Vneo viability, migration and invasion by downregulating PLGF expression.

miR-23b-3p and miR-125b-5p downregulate apo(a) expression by targeting Ets1 in HepG2 cells.

High concentrations of plasma lipoprotein(a) [Lp(a)] have been inferred to be an independent risk factor for cardiovascular and cerebrovascular diseases, such as coronary artery diseases, restenosis, and stroke. Apolipoprotein(a) [apo(a)] is one of the most important components of Lp(a) and contributes greatly to the increased concentration of plasma Lp(a). As a critical positive transacting factor of apo(a) gene, Ets1 has been proven as a target gene of several miRNAs, such as miR-193b, miR-125b-5p, miR-200b, miR-1, and miR-499. In this study, a series of experiments on miRNAs and relative miRNAs inhibitor delivered HepG2 cells were conducted, and two miRNAs that downregulate the apo(a) by targeting the 3'-UTR of Ets1 were identified. Results showed that apo(a) and Ets1 were differentially expressed in SMMC7721 and HepG2 cell lines. Meanwhile, apo(a) and Ets1 were inversely correlated with several hepatic endogenous miRNAs, such as miR-125b-5p, miR-23b-3p, miR-26a-5p, and miR-423-5p, which were predicted to bind to Ets1. Results show that miR-125b-5p and miR-23b-3p mimics could inhibit the synthesis of apo(a) by directly targeting Ets1 in HepG2, thereby reducing the plasma Lp (a) concentration.

H2 S inhibits apo(a) expression and secretion through PKCα/FXR and Akt/HNF4α pathways in HepG2 cells.

Lipoprotein(a) [Lp(a)] is a strong genetic risk factor for coronary heart diseases. However, the metabolism of this protein remains poorly understood. Efficient and specific drugs that can decrease high plasma levels of Lp(a) have not been developed yet. Hydrogen sulfide (H2 S), a member of the gas transmitter family, performs important biological actions, including protection against cardiovascular diseases and maintenance of the lipid metabolism equilibrium in hepatocytes and adipocytes. In this study, we investigated the possible molecular mechanism of H2 S that influences apolipoprotein(a) [apo(a)] biosynthesis. We also determined the effects of H2 S on apo(a) expression and secretion in HepG2 cells as well as the underlying mechanisms. Results showed that H2 S significantly inhibited the expression and secretion levels of apo(a). These effects were attenuated by the PKCα inhibitor and FXR siRNA. H2 S also reduced HNF4α expression and enhanced FXR expression. The Akt inhibitor partially reversed H2 S-induced inhibition of apo(a) and HNF4α expression and apo(a) secretion. This study reveals that H2 S suppressed apo(a) expression and secretion via the PKCα-FXR and PI3K/Akt-HNF4α pathways.

The possible mechanisms of ferroptosis in sepsis-associated acquired weakness.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection, and its morbidity and mortality rates are increasing annually. It is an independent risk factor for intensive care unit-acquired weakness (ICU-AW), which is a common complication of patients in ICU. This situation is also known as sepsis-associated acquired weakness (SAW), and it can be a complication in more than 60% of patients with sepsis. The outcomes of SAW are often prolonged mechanical ventilation, extended hospital stays, and increased morbidity and mortality of patients in ICUs. The pathogenesis of SAW is unclear, and an effective clinical treatment is not available. Ferroptosis is an iron-dependent type of cell death with unique morphological, biochemical, and genetic features. Unlike other forms of cell death such as autophagy, apoptosis, and necrosis, ferroptosis is primarily driven by lipid peroxidation. Cells undergo ferroptosis during sepsis, which further enhances the inflammatory response. This process leads to increased cell death, as well as multi-organ dysfunction and failure. Recently, there have been sporadic reports suggesting that SAW is associated with ferroptosis, but the exact pathophysiological mechanisms remain unclear. Therefore, we reviewed the possible pathogenesis of ferroptosis that leads to SAW and offer new strategies to prevent and treat SAW.

SUMOylation targeting mitophagy in cardiovascular diseases.

Small ubiquitin-like modifier (SUMO) plays a key regulatory role in cardiovascular diseases, such as cardiac hypertrophy, hypertension, atherosclerosis, and cardiac ischemia-reperfusion injury. As a multifunctional posttranslational modification molecule in eukaryotic cells, SUMOylation is essentially associated with the regulation of mitochondrial dynamics, especially mitophagy, which is involved in the progression and development of cardiovascular diseases. SUMOylation targeting mitochondrial-associated proteins is admittedly considered to regulate mitophagy activation and mitochondrial functions and dynamics, including mitochondrial fusion and fission. SUMOylation triggers mitochondrial fusion to promote mitochondrial dysfunction by modifying Fis1, OPA1, MFN1/2, and DRP1. The interaction between SUMO and DRP1 induces SUMOylation and inhibits lysosomal degradation of DRP1, which is further involved in the regulation of mitochondrial fission. Both SUMOylation and deSUMOylation contribute to the initiation and activation of mitophagy by regulating the conjugation of MFN1/2 SERCA2a, HIF1α, and PINK1. SUMOylation mediated by the SUMO molecule has attracted much attention due to its dual roles in the development of cardiovascular diseases. In this review, we systemically summarize the current understanding underlying the expression, regulation, and structure of SUMO molecules; explore the biochemical functions of SUMOylation in the initiation and activation of mitophagy; discuss the biological roles and mechanisms of SUMOylation in cardiovascular diseases; and further provide a wider explanation of SUMOylation and deSUMOylation research to provide a possible therapeutic strategy for cardiovascular diseases. Considering the precise functions and exact mechanisms of SUMOylation in mitochondrial dysfunction and mitophagy will provide evidence for future experimental research and may serve as an effective approach in the development of novel therapeutic strategies for cardiovascular diseases. Regulation and effect of SUMOylation in cardiovascular diseases via mitophagy. SUMOylation is involved in multiple cardiovascular diseases, including cardiac hypertrophy, hypertension, atherosclerosis, and cardiac ischemia-reperfusion injury. Since it is expressed in multiple cells associated with cardiovascular disease, SUMOylation can be regulated by numerous ligases, including the SENP family proteins PIAS1, PIASy/4, UBC9, and MAPL. SUMOylation regulates the activation and degradation of PINK1, SERCA2a, PPARγ, ERK5, and DRP1 to mediate mitochondrial dynamics, especially mitophagy activation. Mitophagy activation regulated by SUMOylation further promotes or inhibits ventricular diastolic dysfunction, perfusion injury, ventricular remodelling and ventricular noncompaction, which contribute to the development of cardiovascular diseases.

PCSK9 mediates the oxidative low­density lipoprotein­induced pyroptosis of vascular endothelial cells via the UQCRC1/ROS pathway.

The present study aimed to explore the role and mechanisms of proprotein convertase subtilisin/kexin type 9 (PCSK9) in the oxidized low­density lipoprotein (oxLDL)­induced pyroptosis of vascular endothelial cells. For this purpose, human umbilical vein endothelial cells (HUVECs) were incubated with oxLDL (100 µg/ml) for 24 h to induce pyroptosis, which was detected using PI/hoechst33342 double staining. The expression of pyroptosis­associated molecules was measured by western blot analysis and RT­qPCR. Reactive oxygen species (ROS) and membrane potential were examined through ROS probe and JC­1 staining, respectively. PCSK9 and mitochondrial ubiquinol­cytochrome c reductase core protein 1 (UQCRC1) protein were knocked down by small interfering RNA (siRNA). PCSK9 was overexpressed by lentivirus. The results revealed that oxLDL induced HUVEC injury, pyroptosis and inflammatory factor release, and upregulated the expression of PCSK9 protein in the HUVECs in a concentration­dependent manner. The silencing of PCSK9 expression with siRNA suppressed the oxLDL­induced damage to HUVECs, the release of inflammatory substances and the occurrence of pyroptosis. In addition, oxLDL inhibited UQCRC1 expression, promoted mitochondrial membrane potential collapse and damaged mitochondrial function; however, these processes were reversed by the silencing of PCSK9. PCSK9 overexpression induced the pyroptosis of HUVECs, the generation of ROS and the disorder of mitochondrial function by inhibiting UQCRC1. Therefore, PCSK9 mediates the oxLDL­induced pyroptosis of vascular endothelial cells via the UQCRC1/ROS pathway.

Inhibition of the ox-LDL-Induced Pyroptosis by FGF21 of Human Umbilical Vein Endothelial Cells Through the TET2-UQCRC1-ROS Pathway.

Fibroblast growth factor 21 (FGF21) is a hormone-like member of the FGF family that is associated with cell death in atherosclerosis. However, its underlying mechanisms remain unclear. In this study, the effect of FGF21 on endothelial cell pyroptosis and its potential mechanisms were investigated. Results showed that FGF21 inhibits oxidized low-density lipoprotein (ox-LDL)-induced pyroptosis and related molecular expression in human umbilical vein endothelial cells (HUVECs). Mitochondrial function was damaged by ox-LDL and restored by FGF21. A mechanism proved that ubiquinol cytochrome c reductase core protein I (UQCRC1) was downregulated by ox-LDL and upregulated by FGF21. Further, the silencing of UQCRC1 aggravated HUVEC pyroptosis and impaired mitochondrial function and reactive oxygen species (ROS) production. Moreover, Tet methylcytosine dioxygenase (TET2) was involved in the regulation of UQCRC1 expression and pyroptosis. In summary, FGF21 inhibited ox-LDL-induced HUVEC pyroptosis through the TET2-UQCRC1-ROS pathway.

Vitamin C down-regulate apo(a) expression via Tet2-dependent DNA demethylation in HepG2 cells.

Lipoprotein(a)[Lp(a)] is a risk factor for coronary heart diseases. However, the metabolism of this protein remains poorly understood. Efficient and specific drugs that can decrease high plasma levels of Lp(a) have not been developed yet. Vitamin C is responsible for maintaining the catalytic activity of a group of iron and 2-oxoglutarate (2OG)-dependent dioxygenases and induces the generation of 5-hydroxymethylcytosine (5hmC) via Ten-eleven translocation (Tet) dioxygenases. In addition, It has been reported vitamin C deficiency induces atherosclerosis and increases Lp(a) and apo(a) plasma levels in Lp(a)+ mice. However, the mechanism is still unclear. In this study, we investigated the effects of vitamin C on apo(a) expression and the possible molecular mechanism of vitamin C that influences apolipoprotein(a) [apo(a)] biosynthesis in HepG2 cells. Results showed that vitamin C significantly inhibited the expression and secretion levels of apo(a). Vitamin C can also increase ELK1 expression and hydroxymethylation of ELK1 promoter and the globle DNA in HepG2 cells. In addition, the effects of vitamin C inhibiting the apo(a) expression were attenuated by ELK1siRNA and Tet2siRNA. These results suggested vitamin C down-regulate apo(a) expression via Tet2-dependent DNA demethylation in HepG2 cells.

Ox-Lp(a) transiently induces HUVEC autophagy via an ROS-dependent PAPR-1-LKB1-AMPK-mTOR pathway.

Oxidised lipoprotein(a) [oxLp(a)] is considered as a more potent arteriosclerotic factor than native Lp(a). However, the molecular mechanisms underlying this potency remain unclear. Reactive oxygen species (ROS) possibly act as intracellular second messengers that participate in autophagy stimulation. In this study, the effect of oxLp(a) on endothelial cell autophagy was determined. The mechanism and effect of autophagy on endothelial cells were also investigated. Results showed that oxLp(a) could induce autophagy depending on the generation of cellular ROS. Superoxide dismutase, an antioxidant, could inhibit oxLp(a)-induced autophagy in human umbilical vascular endothelial cells. Furthermore, poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1)-liver kinase B1 (LKB1)-adenosine monophosphate-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) and LKB1-AMPK-mTOR pathways are involved in oxLp(a)-induced autophagy. These pathways are also dependent on ROS. Thus, oxLp(a) induced autophagy via LKB1-AMPK-mTOR and PAPR-1-LKB1-AMPK-mTOR pathways, which are dependent on ROS generation.

FGF21 increases cholesterol efflux by upregulating ABCA1 through the ERK1/2-PPARγ-LXRα pathway in THP1 macrophage-derived foam cells.

FGF21, a member of the fibroblast growth factor superfamily, is an important endogenous regulator of systemic glucose and lipid metabolism. Elevated serum FGF21 levels have been reported in subjects with coronary heart disease and carotid artery plaques. However, whether FGF21 is associated with atherosclerotic diseases remains unclear. In this study, the effects of FGF21 on cholesterol efflux in THP1 macrophage-derived foam cells and the underlying mechanisms were investigated. THP1 macrophage-derived foam cells were incubated with 0, 25, 50, 100, 200, and 400 ng/mL of FGF21 for varying time periods (0, 6, 12, and 24 h). Cholesterol efflux onto apoA-1 was assessed by high-performance liquid chromatography assays, while change in ABCA1 expression was analyzed by western blot and real-time quantitative PCR. Incubation was performed with the ERK1/2-specific inhibitor PD98059, PPARγ-specific inhibitor GW9662, and LXRα siRNA. Our results show that FGF21 promotes cholesterol efflux and ABCA1 expression in THP1 macrophage-derived foam cells in a dose- and time-dependent manner. In addition, inhibition of ERK1/2 or PPARγ, or knockdown of LXRα attenuated FGF21-mediated promotion of ABCA1 expression and cholesterol efflux. These results demonstrate that FGF21 can promote cholesterol efflux by upregulating ABCA1 through the ERK1/2-PPARγ-LXRα pathway in THP1 macrophage-derived foam cells.

Oxidized lipoprotein(a) increases endothelial cell monolayer permeability via ROS generation.

Oxidized lipoprotein(a) (oxLp(a)) is a more potent marker of atherogenesis than native Lp(a). However, the molecular mechanisms of oxLp(a) activity are not clear. Reactive oxygen species (ROS) have recently been suggested as acting as intracellular second messengers. In this study, the effects of oxLp(a) on endothelial cell monolayer permeability and the role of reactive oxygen species (ROS) generation in these effects were investigated. Our results showed that oxLp(a) inhibited desmoglein-1 (DSG1) and desmocollin-2 (DSC2) expression at both mRNA and protein levels in a dose- and time-dependent manner, and increased the generation of cellular ROS. Down-regulation of DSG1 and DSC2 was strengthened by pretreatment with H2O2 and attenuated by superoxide dismutase (SOD) treatment. Furthermore, oxLp(a) increased endothelial cell monolayer permeability, and this effect was enhanced by H2O2 and blunted by SOD. Taken together, these results demonstrate that oxLp(a) increases endothelial cell monolayer permeability, which is mediated at least in part via ROS generation.