Apoptosis Of Human Umbilical Vein Endothelial Cells Research Articles

Maslinic Acid Suppresses High Glucose-induced Inflammation by Epigenetically Inhibiting TXNIP Expression.

Hyperglycemia-induced inflammation and subsequent endothelial injuries ultimately lead to the pathogenesis of cardiovascular diseases associated with high mortality, such as atherosclerosis. Maslinic acid (MA) is a phytochemical with anti-inflammatory activity. However, it remains unknown whether it can inhibit diabetes-associated cardiovascular inflammation. The present study aimed to determine the effect of MA on high glucose-induced endothelial inflammation and apoptosis in human umbilical vein endothelial cells (HUVECs) and to explore the underlying mechanism. HUVECs were treated with high glucose to induce inflammation and apoptosis. Apoptosis was determined by flow cytometry. CCK-8 assay was used to examine cell viability. Production levels of cytokines were detected by quantitative realtime PCR (qPCR) and ELISA. Protein expression levels and signaling pathways activation were detected by Western blotting. RNA immunoprecipitation and qPCR were used to determine the N6-methyladenosine (m6A) levels of target mRNAs. MA promoted the recruitment of RNA demethylase ALKBH5 to TXNIP mRNA, and subsequently enhanced its m6A demethylation. By this means, MA decreased the stability of TXNIP mRNA and downregulated its expression level. Subsequently, reactive oxygen species (ROS) and production of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-1β, were inhibited. And high glucose-induced apoptosis in HUVECs was inhibited by MA. MA ameliorates high glucose-induced endothelial inflammation and injury, serving as a new potential therapeutic application for protecting against diabetes-associated atherosclerosis and other inflammatory diseases.

Astragaloside IV protects against oxidized low-density lipoprotein-induced injury in human umbilical vein endothelial cells via the histone deacetylase 9 (HDAC9)/NF-κB axis.

Atherosclerosis is a main cause of multiple cardiovascular diseases, and cell damage of human umbilical vein endothelial cells (HUVECs) was reported to participate in the development of atherosclerosis. In this study, we aimed to study the action of Astragaloside IV (ASV) on AS development using in vitro AS cell model. MTT assay, EdU staining assay, and flow cytometry were utilized for detection of cell proliferation and apoptosis, respectively. The protein expression of histone deacetylase 9 (HDAC9), Bax, Bcl-2, p-P65, P65, p-IκBα, and IκBα was gaged using western blot. The angiogenesis was evaluated by tube formation assay. The inflammatory response was evaluated by ELISA kits. SOD activity and MDA level were detected using the matched commercial kits. RT-qPCR was used for HDAC9 mRNA expression measurement. Oxidized low-density lipoprotein (ox-LDL) significantly repressed cell proliferation, angiogenesis, and enhanced apoptosis, inflammation, and oxidative stress in HUVECs. ASV addition could alleviate ox-LDL-caused cell damage in HUVECs. Moreover, HDAC9 was overexpressed in AS patients and AS cell model. Functionally, HDAC9 knockdown also exhibited the protective role in ox-LDL-treated HUVECs. In addition, ASV treatment protected against ox-LDL-induced damage in HUVECs via targeting HDAC9. ASV could inactivate the NF-κB pathway via regulating HDAC9 in AS cell model. ASV exerted the protective effects on ox-LDL-induced damage in HUVECs through the HDAC9/NF-κB axis.

Acrolein evokes inflammation and autophagy-dependent apoptosis through oxidative stress in vascular endothelial cells and its protection by 6-C-(E-2-fluorostyryl)naringenin

• Acrolein induced mitochondrial dysfunction, apoptosis, and inflammation in HUVECs. • Acrolein induced apoptosis was partially dependent on the activation of autophagy. • The addition of styryl group contributed to the protective potential of 6- C -( E -fluorostyryl)naringenin. • The protective effects of 6- C -( E -fluorostyryl)naringenin is via Nrf2 activation. Acrolein is a common dietary and environmental toxicant. Evidence suggests that acrolein can increase the risk of cardiovascular diseases in which endothelial cells are the sensitive targets. In human umbilical vein endothelial cells (HUVECs), acrolein treatment induced mitochondrial dysfunction, which subsequently triggered apoptosis and inflammation. Acrolein also upregulated autophagy, which possibly resulted from negative regulation of the protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway via reactive oxygen species (ROS). Inhibition of autophagy by 3-methyladenine reduced acrolein-evoked apoptosis, implying a pro-death role of autophagy. In addition, the protective potential of 6- C -( E -2-fluorostyryl)naringenin (6-CEFN) against acrolein was examined. 6-CEFN exhibited higher efficacy than its parental compound naringenin in preventing acrolein-caused damages, where nuclear factor erythroid 2-related factor (Nrf2)-mediated up-regulation of phase II enzymes was involved. Altogether, our findings offer new mechanistic insights into acrolein-induced endothelial injury and demonstrate the therapeutic potential of 6-CEFN for acrolein-related cardiovascular toxicities.

The KLF4-p62 axis prevents vascular endothelial cell injury via the mTOR/S6K pathway and autophagy in diabetic kidney disease.

Diabetic kidney disease (DKD) is a complication of systemic diabetic microangiopathy, which has a high risk of developing into end-stage renal disease and death. This study explored the mechanism underlying autophagy in DKD vascular endothelial cell injury. DKD and vascular endothelial cell injury models were established using Sprague Dawley rats and human umbilical vein endothelial cells (HUVECs). HUVECs overexpressing Kruppel-like factor 4 (KLF4) were constructed by transient transfection of plasmids. Biochemical determination of urinary protein and blood urea nitrogen (BUN), superoxide dismutase (SOD), and creatinine (Scr) levels was performed. Renal pathology was observed by periodic acid-Schiff (PAS) staining. Cell Counting Kit-8 (CCK8), terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), and immunocytochemistry (ICC) were used to analyse the growth and apoptosis of HUVECs. Microtubule-associated protein light chain 3 (LC3) expression was observed by immunofluorescence (IF). The reactive oxygen species (ROS) levels were measured using flow cytometry. Monocyte chemoattractant protein-1 (MCP-1), KLF4, and tumour necrosis factor alpha (TNF-α) levels were detected using enzyme-linked immunosorbent assay (ELISA). The expression of KLF4, p62 protein, and LC3 was analysed using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). S6 kinase (S6K), p70 ribosomal S6 kinase (p-S6K), Beclin1, ATG5, LC3, p62, Caspase-3, mammalian target of rapamycine (mTOR), and phsophorylated mTOR (p-mTOR) expressions were detected by western blotting. PAS-positive substances (polysaccharide and glycogen) and S6K protein levels increased, and LC3 protein expression decreased in DKD rats. The levels of urinary protein, BUN, and Scr increased, and KLF4 decreased in DKD rats. High glucose (HG) levels decreased the proliferation and increased the apoptosis rate of HUVECs. The expression of ROS, TNF-α, MCP-1, and p62 increased, while the expression of SOD, KLF4, Beclin1, ATG5, and LC3 decreased in HG-induced HUVECs. KLF4 overexpression significantly increased Beclin1, ATG5, and LC3 protein expression and decreased p62 protein expression compared to the oe-NC group in HG-induced HUVECs. KLF4 overexpression inhibits the expression of Caspase-3, p-mTOR, and p-S6K in HG-induced HUVECs. KLF4-p62 axis improved vascular endothelial cell injury by regulating inflammation and the mTOR/S6K pathway in DKD.

Triterpenoid saponins of Ilex pubescens against TNF-α induced inflammation and apoptosis in human umbilical vein endothelial cells via autophagy pathway.

Triterpenoid saponins of Ilex pubescens (IPTS), the main active components of Ilex pubescens, has a therapeutic effect on atherosclerosis (AS). The ingredients in IPTS that could be intracellularly transported by human umbilical vein endothelial cells (HUVECs) may play an essential role in AS. This study attempted to explore its mechanism from the perspectives of HUVECs' inflammation, apoptosis, and autophagy. By using a tumour necrosis factor-α (TNF-α)-induced HUVECs injury model, cell viability and the expression of intercellular adhesion molecule 1 (ICAM1), matrix metalloproteinase 9 (MMP9), cleave-caspase-3 and cleave-caspase-9, in combination with the results of flow cytometry, JC-1 and Hoechst 33258 staining were investigated to evaluate the anti-inflammatory and anti-apoptotic impact effects of IPTS on HUVECs. Afterwards, the expression of microtubule-associated proteins light chain 3II (LC3II) and sequestosome 1 (p62) was determined to test the effect of IPTS on autophagy. Finally, by adding an autophagy inhibitor 3-methyladenine (3-MA), we investigated whether IPTS exerts anti-inflammatory and anti-apoptotic effects through the autophagy pathway. We firstly demonstrated that pretreatment with IPTS could increase the cell viability, maintain the cell morphology and reduce TNF-α-induced inflammation and apoptosis of HUVECs. Moreover, IPTS pretreatment was proved to raise the expression of LC3II /LC3I while decreasing the expression of p62, which indicated that IPTS could activate HUVECs' autophagy. IPTS has been shown for the first time to exert anti-inflammatory and anti-apoptotic effects through autophagy and thereby resisting TNF-α-induced inflammatory injury of HUVECs. This study preliminarily confirmed that IPTS ameliorated HUVECs' inflammation and apoptosis by increasing autophagy.

Diesel exhaust particles induce human umbilical vein endothelial cells apoptosis by accumulation of autophagosomes and caspase-8 activation

Diesel exhaust particles (DEP) are risk factors for endothelial cells (ECs) dysfunction. However, the mechanism by which DEP induce ECs apoptosis remains unclear. Here, we investigated how DEP induce death of human umbilical vein ECs (HUVECs), with a focus on the autophagy-mediated apoptotic pathway. DEP induced dose-dependent HUVECs death and exposure to the IC50 concentration of DEP (70 µg/ml) led to apoptosis. DEP phosphorylated Beclin-1 (Ser93) and increased protein levels of p62 and LC3BII and the number of LC3B puncta, indicating autophagy initiation. DEP increased expression of pro- and mature forms of cathepsin D, which increases lysosomal activity. However, DEP suppressed expression of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins (STX17, VAMP8, SNAP29, YKT6, and STX7) to inhibit autolysosome formation, resulting in accumulation of autophagosomes. LC3B, p62, and caspase-8 form a tertiary complex in accumulated autophagosomes, which is known to serve as a platform for caspase-8 activation. Indeed, DEP activates caspase-8 and pretreatment with a caspase-8 inhibitor suppressed DEP-induced apoptosis. Furthermore, depletion of p62 decreased caspase-8 and caspase-3 activation and inhibited the DEP-induced apoptosis. Taken together, these findings demonstrated that DEP induced HUVECs apoptosis by inhibiting autophagosome maturation and identified caspase-8 as a novel mediator of DEP-induced ECs apoptosis.

Amygdalin attenuates PM2.5-induced human umbilical vein endothelial cell injury via the TLR4/NF-κB and Bcl-2/Bax signaling pathways.

Mounting evidence supports that long-term exposure to fine particle pollutants (PM2.5) is closely implicated in cardiovascular diseases, especially atherosclerosis. Amygdalin is reported to attenuate external stimuli-induced cardiovascular diseases. However, the underlying mechanisms are still not understood. In this study, we aim to explore the protective effects of amygdalin on PM2.5-induced human umbilical vein endothelial cell (HUVEC) injury and unravel the specific mechanisms by MTT, DCFH-DA, biochemical, immunofluorescence, ELISA, RT-qPCR, flow cytometry, TUNEL and western blot analysis. The results reveal that amygdalin reverses PM2.5-induced cytotoxicity and attenuates intracellular ROS production. Moreover, amygdalin increases the levels of SOD and GSH and alleviates the MDA content. Additionally, amygdalin causes a decline of IL-6, IL-1β, TNF-α and COX-2 levels. Moreover, amygdalin inhibits NF-κB p50 and TLR4 protein expressions and NF-κB p65 nuclear translocation. Concomitantly, a decline of phospho-NF-κB p65/NF-κB p65 and phospho-IκB-α/IκB-α is detected. Meanwhile, amygdalin pretreatment reduces HUVEC apoptosis. In addition, amygdalin triggers an upregulation of Bcl-2 and a downregulation of Bax after stimulation with PM2.5. Collectively, these results suggest that amygdalin suppresses PM2.5-induced HUVEC injury by regulating the TLR4/NF-κB and Bcl-2/Bax signaling pathways, indicating that amygdalin may be a novel target for atherosclerosis treatments.

Polydatin reverses oxidation low lipoprotein (oxLDL)-induced apoptosis of human umbilical vein endothelial cells via regulating the miR-26a-5p/BID axis.

Atherosclerosis is a disease in which lipids and inflammatory factors accumulate on the walls of arteries, forming plaques that eventually block the flow of blood. Polydatin was derived from plant knotweed, which could play an important role in inhibiting the progression of atherosclerosis. However, the mechanism by which polydatin regulates the genesis and development of atherosclerosis remains unclear. To detect the function of polydatin in atherosclerosis, the proliferation, apoptosis and migration of human umbilical vein endothelial cells (HUVECs) was detected using 5-ethynyl-2’-deoxyuridine staining, flow cytometry and transwell assays, respectively. In addition, the branch points and capillary length of HUVECs were observed using a tube formation assay, and the lipid accumulation was tested by Oil-red O staining assay. Dual luciferase reporter assays were performed to confirm the association between microRNA (miR)-26a-5p and BH3 interacting domain death agonist (BID) in HUVECs. The data suggested oxidized low-density lipoprotein (oxLDL) notably inhibited the viability of HUVECs in a dose-dependent manner, and polydatin reversed the oxLDL-induced inhibition of HUVECs viability and proliferation. In addition, polydatin inhibited the apoptosis, migration and epithelial mesenchymal transition (EMT) process in oxLDL-treated HUVECs. Polydatin reversed oxLDL-induced lipid accumulation and angiogenesis inhibition in HUVECs. Furthermore, BID was targeted by miR-26a-5p, and polydatin reversed the oxLDL-induced apoptosis of HUVECs via regulating the miR-26a-5p/BID axis. In summary, polydatin reversed the oxLDL-induced apoptosis of HUVECs via regulating the miR-26a-5p/BID axis. Therefore, polydatin could act as a new agent for atherosclerosis treatment.

Effects and mechanisms of 6-hydroxykaempferol 3,6-di-O-glucoside-7-O-glucuronide from Safflower on endothelial injury in vitro and on thrombosis in vivo.

Background: The florets of Carthamus tinctorius L. (Safflower) is an important traditional medicine for promoting blood circulation and removing blood stasis. However, its bioactive compounds and mechanism of action need further clarification. Objective: This study aims to investigate the effect and possible mechanism of 6-hydroxykaempferol 3,6-di-O-glucoside-7-O-glucuronide (HGG) from Safflower on endothelial injury in vitro, and to verify its anti-thrombotic activity in vivo. Methods: The endothelial injury on human umbilical vein endothelial cells (HUVECs) was induced by oxygen-glucose deprivation followed by reoxygenation (OGD/R). The effect of HGG on the proliferation of HUVECs under OGD/R was evaluated by MTT, LDH release, Hoechst-33342 staining, and Annexin V-FITC apoptosis assay. RNA-seq, RT-qPCR, Enzyme-linked immunosorbent assay and Western blot experiments were performed to uncover the molecular mechanism. The anti-thrombotic effect of HGG in vivo was evaluated using phenylhydrazine (PHZ)-induced zebrafish thrombosis model. Results: HGG significantly protected OGD/R induced endothelial injury, and decreased HUVECs apoptosis by regulating expressions of hypoxia inducible factor-1 alpha (HIF-1α) and nuclear factor kappa B (NF-κB) at both transcriptome and protein levels. Moreover, HGG reversed the mRNA expression of pro-inflammatory cytokines including IL-1β, IL-6, and TNF-α, and reduced the release of IL-6 after OGD/R. In addition, HGG exhibited protective effects against PHZ-induced zebrafish thrombosis and improved blood circulation. Conclusion: HGG regulates the expression of HIF-1α and NF-κB, protects OGD/R induced endothelial dysfunction in vitro and has anti-thrombotic activity in PHZ-induced thrombosis in vivo.

The combination of EGCG with warfarin reduces deep vein thrombosis in rabbits through modulating HIF-1α and VEGF via the PI3K/AKT and ERK1/2 signaling pathways.

Deep venous thrombosis (DVT) poses a major challenge to public health worldwide. Endothelial cell injury evokes inflammatory and oxidative responses that contribute to thrombus formation. Tea polyphenol (TP) in the form of epigallocatechin-3-gallate (EGCG) has anti-inflammatory and oxidative effect that may ameliorate DVT. However, the precise mechanism remains incompletely understood. The current study was designed to investigate the anti-DVT mechanism of EGCG in combination with warfarin (an oral anticoagulant). Rabbits were randomly divided into five groups. A DVT model of rats was established through ligation of the inferior vena cava (IVC) and left common iliac vein, and the animals were orally administered with EGCG, warfarin, or vehicle for seven days. In vitro studies included pretreatment of human umbilical vein endothelial cells (HUVECs) with different concentrations of EGCG for 2 h before exposure to hydrogen peroxide. Thrombus weight and length were examined. Histopathological changes were observed by hematoxylin-eosin staining. Blood samples were collected for detecting coagulation function, including thrombin and prothrombin times, activated partial thromboplastin time, and fibrinogen levels. Protein expression in thrombosed IVCs and HUVECs was evaluated by Western blot, immunohistochemical analysis, and/or immunofluorescence staining. RT-qPCR was used to determine the levels of AGTR-1 and VEGF mRNA in IVCs and HUVECs. The viability of HUVECs was examined by CCK-8 assay. Flow cytometry was performed to detect cell apoptosis and ROS generation was assessed by 2',7'-dichlorofluorescein diacetate reagent. In vitro and invivo studies showed that EGCG combined with warfarin significantly reduced thrombus weight and length, and apoptosis in HUVECs. Our findings indicated that the combination of EGCG and warfarin protects HUVECs from oxidative stress and prevents apoptosis. However, HIF-1α silencing weakened these effects, which indicated that HIF-1α may participate in DVT. Furthermore, HIF-1α silencing significantly up-regulated cell apoptosis and ROS generation, and enhanced VEGF expression and the activation of the PI3K/AKT and ERK1/2 signaling pathways. In conclusion, our results indicate that EGCG combined with warfarin modifies HIF-1α and VEGF to prevent DVT in rabbits through anti-inflammation via the PI3K/AKT and ERK1/2 signaling pathways.

Circ_0003423 Alleviates Oxidized Low-Density Lipoprotein-Induced Endothelial Cell Injury by Sponging miR-142-3p and Activating Sirtuin 3/Superoxide Dismutase 2 Pathway.

Atherosclerosis (AS) is a common cardiovascular disease with high morbidity and mortality globally. Circular RNAs (circRNAs) have been shown to regulate AS progression. However, the biological role of circ_0,003,423 in AS pathology and its associated mechanism is still unclear. Oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) were used as an AS cell model invitro. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assays were conducted to measure ribonucleic acid (RNA) and protein expression. Cell proliferation was analyzed by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays. Cell apoptosis, migration, and angiogenesis were analyzed by flow cytometry, wound healing, and Matrigel tube formation assays. The oxidative stress factors were detected using reactive oxygen species activity (ROS) assay kit, malondialdehyde (MDA) assay kit, and superoxide dismutase (SOD) assay kit. The interaction between microRNA-142-3p (miR-142-3p) and circ_0,003,423 or sirtuin 3 (SIRT3) was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Ox-LDL exposure dose-dependently reduced circ_0,003,423 expression in HUVECs. Ox-LDL suppressed the proliferation, migration, and angiogenesis and induced the apoptosis and oxidative stress of HUVECs, which were offset by circ_0,003,423 overexpression. miR-142-3p is a direct target of circ_0,003,423, and circ_0,003,423 overexpression-mediated protective effects in ox-LDL-induced HUVECs were largely reversed by the addition of miR-142-3p mimic. miR-142-3p directly interacted with the 3'UTR of SIRT3 and SIRT3 knockdown reversed anti-miR-142-3p-mediated protective effects in HUVECs upon ox-LDL exposure. Circ_0,003,423 positively regulated SIRT3 expression by sponging miR-142-3p in HUVECs. In addition, circ_0,003,423 could regulate the SIRT3/SOD2 signaling pathway in HUVECs. Circ_0,003,423 protected HUVECs from ox-LDL-induced dysfunction by sponging miR-142-3p and activating SIRT3/SOD2 signaling, which provided new potential targets for AS intervention and treatment.

MiR-125a-3p aggravates ox-LDL-induced HUVEC injury through BAMBI.

Atherosclerosis (AS) is a chronic inflammatory disease characterized by the formation of atherosclerotic plaque in the intima of arteries. Among the known regulators of atherosclerosis, microRNAs (miRNAs) have been reported to play critical roles in lipoprotein homeostasis and plaque formation. But the roles of microRNA-125a-3p (miR-125a-3p) in the pathogenesis of AS remain unknown. Human umbilical vein endothelial cells (HUVECs) were treated with oxidized low-density lipoprotein (ox-LDL) to construct the vascular injury model of AS pathogenesis in vitro. miR-125a-3p and BMP and activin membrane-bound inhibitor (BAMBI) expression levels in HUVECs were then measured by quantitative real-time polymerase chain reaction and western blot. The viability and apoptosis of HUVECs were analyzed by Cell Counting Kit-8 assay, TUNEL assay, and flow cytometry, respectively. The relationship between BAMBI 3'-untranslated region and miR-125a-3p was validated by dual luciferase reporter gene assay. miR-125a-3p expression was raised in HUVECs induced with ox-LDL. In HUVECs, miR-125a-3p enhanced the effects of ox-LDL treatment on repressing the viability and promoting the apoptosis of cells. Additionally, BAMBI was confirmed as a direct target of miR-125a-3p and BAMBI overexpression reversed the effects of miR-125a-3p on HUVECs. miR-125a-3p aggravates the dysfunction of HUVECs induced by ox-LDL via BAMBI, which implies that miR-125a-3p is involved in the pathogenesis of AS.

Ameliorative effect of dandelion (Taraxacum officinale) peptides on benzo(a)pyrene-induced oxidative stress and inflammation in human umbilical vein endothelial cells.

Dandelion (Taraxacum officinale) is widely consumed as a health food and a traditional medicine. However, the protective effect of dandelion bio-active peptides (DPs) against polycyclic aromatic hydrocarbon-induced blood vessel inflammation and oxidative damage is not well documented. In the current study, four novel DPs were isolated using an activity tracking method. The protective activity of the DPs against benzo(a)pyrene (Bap)-induced human umbilical vein endothelial cell (HUVEC) damage was explored. The results indicated that DP-2 [cycle-(Thr-His-Ala-Trp)] effectively inhibited Bap-induced reactive oxygen species (ROS) and malondialdehyde (MDA) overproduction and reinforced antioxidant enzyme activity while inhibiting the production of inflammatory factors in HUVECs. Moreover, DP-2 increased NAD(P)H:quinone oxidoreductase 1, heme oxygenase-1, and nuclear factor E2-releated factor 2 expression levels by activating the PI3K/Akt signaling pathway. In addition, DP-2 attenuated Bap-induced HUVEC apoptosis via the Bcl-2/Bax/cytochrome c apoptotic pathway. These results suggest that DP-2 is a promising compound for protecting HUVECs from Bap-induced inflammatory and oxidative damage.

The role of PPARγ in intermittent hypoxia-related human umbilical vein endothelial cell injury.

Patients with obstructive sleep apnoea (OSA) have a high incidence of vascular endothelial injury. The most important pathophysiological feature of OSA is chronic intermittent hypoxia (CIH). This study aimed to investigate the mechanisms of CIH-related vascular endothelial injury. IH exposure was applied to human umbilical vein endothelial cells (HUVECs). After modeling, cell viability, the expression levels of peroxisome proliferator activated receptor γ (PPARγ), apoptosis-associated proteins and mitochondrial division fusion proteins, and the levels of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were assessed via Cell Counting Kit-8 (CCK-8), western blotting, fluorescent microscope, and flow cytometry, respectively. Rosiglitazone (PPARγ agonist), tempo (the mitochondrial-specific antioxidant), and tempo combined with PPARγ interfering RNA were used to treat HUVECs, respectively. After IH exposure, cell viability and levels of MMP decreased, cell apoptosis and ROS levels increased, and the expression levels of PPARγ decreased. Both tempo and rosiglitazone pretreatment ameliorated cell apoptosis and improved cell viability. In addition, mitochondrial function became better after tempo pretreatment. PPARγ interference reversed the protective effects of tempo on IH-related mitochondrial function injury and cell injury. PPARγ regulated the apoptosis and cell viability of IH-treated HUVECs by altering mitochondrial function. This finding clarifies the mechanism of CIH-related vascular endothelial injury.

Flavonoids from Lycium barbarum Leaves Exhibit Anti-Aging Effects through the Redox-Modulation.

Lycium barbarum leaves are a kind of vegetable, and modern nutrition studies have found that they have an anti-aging function. Our study aims to investigate the anti-aging effects of Lycium barbarum leaf flavonoid (LBLF) extracts and its underlying molecular mechanism. LBLFs were purified using D101 and polyamide resin, characterized by ultraperformance liquid chromatography coupled with mass spectrometry, and administered to hydrogen peroxide (H2O2)-treated human umbilical vein endothelial cells (HUVECs) and Caenorhabditis elegans. Appropriate enrichment conditions were optimized through dynamic adsorption and desorption experiments, the content of flavonoids reached 909.84 mg/g, rutin and kaempferol being the main ones. LBLFs attenuated H2O2-induced HUVEC apoptosis, decreased reactive oxygen species and malondialdehyde production levels, increased superoxide dismutase, glutathione peroxidase and catalase activities. Furthermore, pre-treatment with LBLF increased mRNA expression of erythropoietin (EPO) and heme oxygenase-1 (HO-1) via the mitogen-activated protein kinase (MAPK) signaling pathway in HUVECs. Compared with 100 µM rutin monomer, LBLF prolonged the lifespan of Caenorhabditis elegans, enhanced their mobility in middle life stages and upregulated expression of sod-2, gcs-1 and skn-1 genes, which indicated that the anti-aging effects of LBLF were due to its redox-modulation.

Ferulic Acid Protects Endothelial Cells from Hypoxia-Induced Injury by Regulating MicroRNA-92a.

Materials and Methods CCK-8 (cell counting kit-8), Western blotting and Annexin V-FITC/PI staining were used to detect cell viability and apoptosis after hypoxia stimulation. The level of microRNA-92 a (miR-92 a) was detected by qRT-PCR.. Then, the assays of flow cytometry and the annexin V/PI staining kit were applied to value the impact of FA on hypoxia-induced cell proliferation, cell cycle distribution, and apoptosis. Furthermore, the inhibitor and mimic of miR-92a were also administrated to explore the role of miR-92a in this process. Student's t-test was used to explore the differences between two groups, while one-way analysis of variance (ANOVA) was used to explore the differences between more than two groups. Results The results showed that hypoxia stimulation significantly inhibited HUVEC viability and proliferation, such as remarkably decreasing the expression of CDK2, CDK4, and cyclin D1 in HUVECs. The results of annexin V-FITC/PI apoptosis detection showed that hypoxia culture significantly induced HUVEC apoptosis, which indicated that hypoxia stimulation significantly inhibited viability and proliferation of HUVECs but caused cell apoptosis and the expression of miR-92a. Meanwhile, FA remarkably protected HUVECs from hypoxia-induced inhibition of viability and proliferation, as well as the enhancement of apoptosis and miR-92a expression. Furthermore, suppression of miR-92a enhanced the protective effects of FA on hypoxia-induced HUVECs, while activation of miR-92a reversed those effects. Conclusion Our study reported that FA preserved HUVECs from hypoxia-induced injury via regulating miR-92a, which facilitated the understanding of the protective capacity of FA in hypoxia-caused HUVEC injury.

Curcumin activates autophagy and attenuates high glucose-induced apoptosis in HUVECs through the ROS/NF-κB signaling pathway.

Curcumin (CUR) is well known for its anti-inflammatory and antioxidant effects. However, the endothelial protective effect of CUR in diabetes and the underlying signaling pathway remains unclear. The goal of the current study was to provide evidence regarding the protective mechanism of CUR against the high glucose (HG)-induced damage to human umbilical vein endothelial cells (HUVECs). HG-induced HUVECs injury model was used to evaluate the protective effect and the underlying mechanism of CUR against endothelial injury. The cell viability was determined by the MTT method. The cell reactive oxygen species (ROS) were determined by using flow cytometry. The protein expression levels of Bcl-2, Bax, LC3-II/I, Beclin-1, p62, cleaved caspase-3, IκBα and NF-κB were measured by the western blotting. Results showed that CUR significantly decreased the cell apoptosis, the ROS generation and the inflammatory cytokine NF-κB activity in the HG-induced HUVECs versus the control, P<0.05. In addition, CUR significantly increased the expressions of LC3-II/I, Beclin-1, IκBα and Bax/Bcl-2 in the HG-induced HUVECs versus the control, P<0.05. Furthermore, the addition of autophagy inhibitor 3-MA impaired the autophagy, exacerbated the apoptotic death and increased the ROS and NF-κB levels in HUVECs under the high glucose condition, P<0.05. In brief, autophagy served a protective role in the HG-induced apoptosis in HUVECs and CUR alleviated apoptosis by promoting autophagy and inhibiting the ROS/NF-κB signaling pathway.

Phoenixin-14 alleviates inflammatory smooth muscle cell-induced endothelial cell dysfunction in vitro

BackgroundIntracranial aneurysm (IA) is cerebrovascular disorder which refers to local vessel wall damage to intracranial arteries, forming abnormal bulge. Both endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are closely associated with IA formation and rupture. Inflammatory SMCs (iSMCs) were reported to induce EC dysfunction and result in IA progression. Phoenixin-14 (PNX-14) is a recently discovered brain peptide with pleiotropic roles, which participates in reproduction, cardio protection, lipid deposition and blood glucose metabolism. PNX-14 was previously reported to protect brain endothelial cells against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced cell injury. Therefore, our study was designed to investigate the influence of PNX-14 on iSMCs-induced endothelial dysfunction. MethodsInflammation in SMCs was induced by cyclic mechanical stretch. Human umbilical vein endothelial cells (HUVECs) were exposed to SMC- or iSMC-conditioned medium and then treated with 100 nM PNX-14 for 24 h. The levels of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) in cell supernatants were analyzed by ELISA. Cell viability, apoptosis, angiogenesis and migration were subjected to CCK-8 assay, flow cytometry analysis, tube formation assay and Transwell migration assay. The protein levels of proinflammatory cytokines and apoptosis markers (Bcl-2 and Bax) were evaluated by western blotting. ResultsCyclic mechanical stretch upregulated IL-1β, IL-6 and TNF-α levels in SMCs. Treatment with SMC- or iSMC-conditioned medium HUVECs inhibited cell viability, angiogenesis and migration and induced apoptosis in HUVECs. iSMC-conditioned medium has more significant effects on cell functions. However, the influence of SMC- or iSMC-conditioned medium treatment on HUVEC biological functions were reversed by PNX-14 treatment. PNX-14 exerts no significant influence on the biological functions of HUVECs treated with SMC medium. ConclusionPNX-14 alleviates iSMCs-induced endothelial cell dysfunction in vitro.

EA.hy926 Cells and HUVECs Share Similar Senescence Phenotypes but Respond Differently to the Senolytic Drug ABT-263.

Doxorubicin (DOX) induces endothelial cell (EC) senescence, which contributes to endothelial dysfunction and cardiovascular complications. Senolytic drugs selectively eliminate senescent cells to ameliorate senescence-mediated pathologies. Previous studies have demonstrated differences between immortalized and primary EC models in some characteristics. However, the response of DOX-induced senescent ECs to senolytics has not been determined across these two models. In the present work, we first established a comparative characterization of DOX-induced senescence phenotypes in immortalized EA.hy926 endothelial-derived cells and primary human umbilical vein EC (HUVECs). Thereafter, we evaluated the senolytic activity of four senolytics across both ECs. Following the DOX treatment, both EA.hy926 and HUVECs shared similar senescence phenotypes characterized by upregulated senescence markers, increased SA-β-gal activity, cell cycle arrest, and elevated expression of the senescence-associated secretory phenotype (SASP). The potentially senolytic drugs dasatinib, quercetin, and fisetin demonstrated a lack of selectivity against DOX-induced senescent EA.hy926 cells and HUVECs. However, ABT-263 (Navitoclax) selectively induced the apoptosis of DOX-induced senescent HUVECs but not EA.hy926 cells. Mechanistically, DOX-treated EA.hy926 cells and HUVECs demonstrated differential expression levels of the BCL-2 family proteins. In conclusion, both EA.hy926 cells and HUVECs demonstrate similar DOX-induced senescence phenotypes but they respond differently to ABT-263, presumably due to the different expression levels of BCL-2 family proteins.

ALKBH5 inhibits TNF-α-induced apoptosis of HUVECs through Bcl-2 pathway

The dysfunction and apoptosis of vascular endothelial cells are the initiating links in the formation of atherosclerosis. N6-methyladenosine (m6A) is an extremely extensive RNA methylation modification and its abnormality leads to the occurrence of various human diseases. In this study, we explored the effects of demethylase α-ketoglutarate-dependent dioxygenase ALKB homolog 5 (ALKBH5) on TNF-α-induced apoptosis of human umbilical vein endothelial cells (HUVECs). In TNF-α-treated HUVECs, the expression of ALKBH5 was significantly decreased. ALKBH5 overexpression promoted the proliferation and inhibited the apoptosis in TNF-α-treated HUVECs, suggesting that ALKBH5 had a protective effect on cell damage induced by TNF-α. Importantly, ALKBH5 promoted the expression of Bcl-2 in HUVECs. Bcl2 overexpression reduced the expression of Gadd45, Bax, and p21, which are transcriptionally activated by p53. But the expression of p53 has not been significantly affected, indicating that Bcl2 might regulate the apoptosis by inhibiting p53 downstream targets. In addition, ALKBH5 overexpression significantly increased the level of pri-miR-7 and decreased the level of miR-7. In conclusion, ALKBH5 attenuated the TNF-α-induced cell injury via promoting Bcl2 expression. Our research expands the understanding of the progression mechanism of atherosclerosis and provides a potential strategy for the protection of vascular endothelial injury.