Oxidative Stress In Human Umbilical Vein Endothelial Cells Research Articles

1-Deoxynojirimycin Attenuates High-Glucose-Induced Oxidative DNA Damage via Activating NRF2/OGG1 Signaling

1-Deoxynojirimycin (DNJ) is a type of alkaloid that mainly exists in mulberry fruit and leaves. DNJ inhibits α-glucosidase, reduces the absorption of sugar, and suppresses after-meal hyperglycemia. It was reported that DNJ functions in attenuating cellular oxidative stress. However, the mechanisms remain largely unknown. In this study, we firstly confirmed that 5 µmol/L DNJ treatment mitigated the oxidative DNA damage and cell senescence in human umbilical vein endothelial cells (HUVEC) cultured in medium containing 50 mmol/L glucose. Next, we found that DNJ treatment stimulates the expression of anti-oxidative response regulator, Nuclear factor (erythroid-derived 2)-like 2 (NRF2) by around 50% in cells cultured with high glucose. In addition, 8-oxoguanine DNA glycosylase (OGG1) was upregulated by over 15% after DNJ treatment to mitigate high-glucose-induced oxidative DNA damage, and it was identified as a downstream target of NRF2. Further, DNJ treatment promoted the phosphorylation and activation of AKT (ser473) by around 50% in cells cultured with high glucose, and AKT inhibitor treatment abrogated DNJ-induced upregulation of NRF2 and OGG1. Taken together, our results indicate that DNJ is an effective natural antioxidant in mitigating high-glucose-induced oxidative stress in HUVEC via activating the AKT-NRF2-OGG1 anti-oxidative response.

MiR-497-5p regulates ox-LDL-induced dysfunction in vascular endothelial cells by targeting VEGFA/p38/MAPK pathway in atherosclerosis

BackgroundThe impairment of endothelial cells triggered by oxidized low-density lipoprotein (ox-LDL) stands as a critical event in the advancement of atherosclerosis (AS). MiR-497-5p has been recognized as a potential predictor for AS, but its precise involvement in ox-LDL-induced endothelial cell dysfunction remains to be elucidated. MethodsAn in vitro AS cell model was established by exposing human umbilical vein endothelial cells (HUVECs) to 100 μg/mL ox-LDL for 24 h. The assessment of endothelial cell function included evaluating cell viability, caspase-3 activity, inflammatory factors, and oxidative markers. Molecular mechanisms were elucidated through quantitative real-time PCR, Western blot analysis, and luciferase reporter assays. ResultsOur investigation revealed that exposure to ox-LDL led to an upregulation in miR-497-5p and p-p38 levels, while downregulating the expression of vascular endothelial growth factor A (VEGFA) and phosphorylated (p)-endothelial nitric oxide synthase (p-eNOS) in HUVECs. Ox-LDL exposure resulted in decreased cell viability and angiogenic capacity, coupled with increased apoptosis, inflammation, and oxidative stress in HUVECs, partially mediated by the upregulation of miR-497-5p. We confirmed VEGFA as a direct target of miR-497-5p. Interfering with VEGFA expression significantly reversed the effects mediated by miR-497-5p silencing in HUVECs exposed to ox-LDL. ConclusionsIn summary, our findings demonstrate that miR-497-5p exacerbates ox-LDL-induced dysfunction in HUVECs through the activation of the p38/MAPK pathway, mediated by the targeting of VEGFA.

Notoginsenoside-R1 ameliorates palmitic acid-induced insulin resistance and oxidative stress in HUVEC via Nrf2/ARE pathway.

Panax notoginseng, a Chinese traditional food and herb medicine, possesses notable cardiovascular health-promoting properties, with notoginsenoside (NG)-R1 being a key active compound. Insulin resistance represents a global health concern associated with various metabolic disorders. This study investigated the effects of NG-R1 on palmitic acid (PA)-induced insulin resistance and oxidative stress in human umbilical vein endothelial cells (HUVECs). Our findings demonstrate that NG-R1 significantly alleviated impaired glucose uptake, enhanced the phosphorylation of protein kinase B (PKB/Akt) at Ser473, and reduced the phosphorylation of insulin receptor substrate 1 (IRS-1) at Ser307 in PA-treated HUVECs. Furthermore, NG-R1 treatment significantly lowered the levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), while increasing the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG). Additionally, NG-R1 activated the Nrf2/ARE signaling pathway, leading to a substantial increase in the expression of antioxidant enzymes. Notably, knockdown of Nrf2 attenuated the beneficial effects of NG-R1 on PA-induced insulin resistance and oxidative stress in HUVECs, suggesting that NG-R1 exerts its effects through the Nrf2/ARE pathway. In summary, our study reveals that NG-R1 ameliorated PA-induced insulin resistance in HUVECs via Nrf2/ARE pathway, providing novel insights into its potential for alleviating metabolic disorders and cardiovascular disease.

CTRP13 alleviates palmitic acid-induced inflammation, oxidative stress, apoptosis and endothelial cell dysfunction in HUVECs

C1q/tumor necrosis factor-related protein 13 (CTRP13) has been reported to participate in cardiovascular diseases. However, the role and molecular mechanism of CTRP13 in obesity-induced endothelial cell damage is still unclear. In palmitic acid (PA)-induced human umbilical vein endothelial cells (HUVECs), qRT-PCR and western blot were used to examine CTRP13 expression. CCK-8 and TUNEL assays were adopted to assess cell viability and apoptosis, respectively. ROS level and MDA content were evaluated by their commercial kits and inflammatory cytokines were measured using ELISA. Endothelial cell dysfunction was evaluated by detecting NO production and eNOS expression, and tube formation assay was performed to assess angiogenesis. AMPK pathway-related proteins were detected by western blot. The results showed that CTRP13 was downregulated in PA-induced HUVECs. CTRP13 overexpression reduced PA-induced cell viability loss and oxidative stress in HUVECs. Moreover, CTRP13 overexpression suppressed PA-induced inflammation and apoptosis, improved angiogenesis ability, and alleviated endothelial cell dysfunction in HUVECs. In addition, CTRP13 overexpression activated AMPK pathway and regulated the expressions of downstream NOX1/p38 and KLF2. Furthermore, compound C countervailed the impacts of CTRP13 overexpression on cell viability, oxidative stress, inflammation, apoptosis and endothelial function in PA-induced HUVECs. To sum up, CTRP13 overexpression may alleviate PA-induced endothelial cell damage.

ADAMTS13 inhibits H2O2-induced human venous endothelial cell injury to attenuate deep-vein thrombosis by blocking the p38/ERK signaling pathway.

Deep vein thrombosis (DVT) is a common complication in hematologic malignancies and immunologic disorders. Endothelial cell injury and dysfunction comprise the critical contributor for the development of DVT. A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), a plasma metalloprotease that cleaves von Willebrand factor, acts as a critical regulator in normal hemostasis. This study was aimed to explore the role of ADAMTS13 in endothelial cell injury during DVT and the possible mechanism. First, human umbilical vein endothelial cells (HUVECs) were exposed to hydrogen peroxide (H2O2). Then, the mRNA and protein expressions of ADAMTS13 were evaluated with the reverse transcription-quantitative polymerase chain reaction and western blot. After treatment with recombinant ADAMTS13 (rADAMTS13; rA13), the viability and apoptosis of H2O2-induced HUVECs were assessed by cell counting kit-8 assay and terminal-deoxynucleoitidyl transferase-mediated nick end labeling staining. In addition, the levels of prostaglandin F1-alpha, endothelin-1, and reactive oxygen species were detected using the enzyme-linked immunosorbent assay and dichloro-dihydro-fluorescein diacetate assay. The expressions of proteins related to p38/extracellular signal-regulated kinase (ERK) signaling pathway were estimated with the western blot. Then, p79350 (p38 agonist) was used to pretreat cells to analyze the regulatory effects of rA13 on p38/ERK signaling in H2O2-induced HUVEC injury. The results revealed that ADAMTS13 expression was significantly downregulated in H2O2-induced HUVECs. The reduced viability and increased apoptosis of HUVECs induced by H2O2 were revived by ADAMTS13. ADAMTS13 also suppressed the oxidative stress in HUVECs after H2O2 treatment. Besides, ADAMTS13 was found to block p38/ERK signaling pathway, and p79350 reversed the impacts of ADAMTS13 on the damage of HUVECs induced by H2O2. To sum up, ADAMTS13 could alleviate H2O2-induced HUVEC injury through the inhibition of p38/ERK signaling pathway.

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.

H2O2 down-regulates SIRT7's protective role of endothelial premature dysfunction via microRNA-335-5p.

Endothelial senescence is believed to constitute the initial pathogenesis of the atherosclerotic cardiovascular disease (ASCVD). MicroRNA-335-5p (miR-335-5p) expression is significantly up-regulated in oxidative stress-induced endothelial cells (ECs). Sirtuin7 (SIRT7) is considered to prevent EC senescence, yet data on its response to ASCVD risk factors are limited. The present study analyzed the elevated levels of miR-335-5p and the decreased levels of SIRT7 in human umbilical vein endothelial cells (HUVECs), and found that high glucose, tumor necrosis factor-α (TNF-α), and H2O2 are the three contributing factors that induced cellular senescence. The current study also assessed premature endothelial senescence and decreased proliferation, adhesion, migration, and nitric oxide (NO) secretion in HUVECs with these risk factors together with SIRT7–siRNA transfection. It found that the miR-335-5p inhibitor attenuated the down-regulation of SIRT7 expression induced by oxidative stress in HUVECs, and SIRT7 overexpression exerts a rescue effect against miR-335-5p-induced endothelial dysfunction. Furthermore, the direct binding of miR-335-5p to SIRT7 was observed in human embryonic kidney cells 293T (HEK 293T). Therefore, it can be inferred that miR-335-5p down-regulates the expression of SIRT7 in human cells. Current findings may provide deeper insights into the underlying mechanisms of endothelial senescence and potential therapeutic targets of ASCVD as well as other age-related diseases.

Design of biodegradable 3D-printed cardiovascular stent

A 3D cardiovascular stent based on the combination of 2-hydroxy ethyl methacrylate (HEMA) and methacrylate graphene oxide (MeGO) and loaded with resveratrol (RSL) was developed to enhance the mechanical properties and efficiency in recovery of endothelial cell function. A 3D stent model based on the combination of HEMA-MeGO was printed through the extrusion method conducted at a speed of 2 mm/s with a layer height of 0.16 mm, 100% infill density with a 27-gauge needle in a Carbopol support bath. The corresponding rheological properties of HEMA-MeGO hydrogel inks and the mechanical properties of 3D-printed stents were examined. The effects of RSL (∼1 mM) loaded in 3D stents on viability, nitric oxide (NO) production, and downregulation of oxidative stress from the human umbilical vein endothelial cells (HUVECs) were evaluated using MTS, Griess, and DCFDA assays, respectively. The percentage volume change of stents after 24 h made of either HEMA or HEMA-0.35MeGO were 36.7 ± 4.7% and 7.7 ± 1.6%, respectively. The remaining fractions of 3D stents after 21 days and corresponding first-order degradation rate were 95.9 ± 0.5% and 0.30 ± 0.10 × 10−4/hr for HEMA, whereas those were 98.2 ± 0.3% and 0.11 ± 0.04 × 10−4/hr for HEMA-0.35MeGO. The addition of MeGO significantly enhanced the stiffness of hydrogel inks for 3D stents, displaying the Young's modulus of 2.2 ± 0.1 kPa and 4.1 ± 0.02 kPa for HEMA and HEMA-0.35MeGO (F-ratio = 2.99 and p-value < 0.001 for 21 days), respectively. MTS assay revealed that 3D stent loaded with RSL (∼1 mM) exerted no cytotoxic effects on the HUVECs. The controlled release of RSL from the stent enhanced NO production, lowered TNF-α level, and alleviated H2O2-induced oxidative stress in HUVECs. The 3D-printed biodegradable stent based on HEMA-MeGO and loaded with RSL displayed excellent biocompatibility and mechanical properties. RSL released from 3D cardiovascular stents efficiently recovered the endothelial function.

HIF-1\u03b1/JMJD1A signaling regulates inflammation and oxidative stress following hyperglycemia and hypoxia-induced vascular cell injury

BackgroundEndothelial cell (EC) injury accelerates the progression of diabetic macrovascular complications. Hypoxia is an important cause of EC injury. Hypoxia-inducible factor-1 alpha (HIF-1α) is an important hypoxia regulatory protein. Our previous studies showed that high-glucose and hypoxic conditions could upregulate HIF-1α expression and enhance EC inflammatory injury, independently of the nuclear factor kappa-B (NF-κB) pathway. However, it is not clear whether HIF-1α plays a role in vascular disease through epigenetic-related mechanisms.MethodsWe conducted gene expression analysis and molecular mechanistic studies in human umbilical vein endothelial cells (HUVECs) induced by hyperglycemia and hypoxia using RNA sequencing (RNA-seq) and small interfering HIF-1α (si-HIF-1α). We determined HIF-1α and Jumonji domain-containing protein 1 A (JMJD1A) expression by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot, analyzed inflammatory protein secretion in the cell supernatant by enzymelinked immunosorbent assay (ELISA), and assessed protein interaction between HIF-1α and JMJD1A by chromatin immunoprecipitation (Ch-IP). We used the Cell Counting Kit8 (CCK-8) assay to analyze cell viability, and assessed oxidative stress indicators by using a detection kit and flow cytometry.ResultsHigh glucose and hypoxia up-regulated HIF-1α expression, and down-regulated HIF-1α decreased the level of inflammation and oxidative stress in HUVECs. To determine the downstream pathways, we observed histone demethylases genes and related pathway by RNA-sEq. Among these, JMJD1A was the most upregulated gene in histone demethylases. Moreover, we observed that HIF-1α bound to the promoter of JMJD1A, and the ameliorative effects of si-HIF-1α on oxidative stress and inflammatory cytokines in high-glucose and hypoxia-induced HUVECs were reversed by JMJD1A overexpression. Furthermore, knockdown of JMJD1A decreased inflammatory and oxidative stress injury. To determine the JMJD1A-related factors, we conducted gene expression analysis on JMJD1A-knockdown HUVECs. We observed that downregulation of inflammation and the oxidative stress pathway were enriched and FOS and FOSB might be important protective transcription factors.ConclusionsThese findings provide novel evidence that the HIF-1α/JMJD1A signaling pathway is involved in inflammation and oxidative stress in HUVECs induced by high glucose and hypoxia. Also, this pathway might act as a novel regulator of oxidative stress and inflammatory-related events in response to diabetic vascular injury and thus contribute to the pathological progression of diabetes and vascular disease.

Knockdown of Circular Ubiquitin-specific Peptidase 9 X-Linked Alleviates Oxidized Low-density Lipoprotein-induced Injuries of Human Umbilical Vein Endothelial Cells by Mediating the miR-148b-3p/KLF5 Signaling Pathway.

There is evidence that the development of atherosclerosis (AS) involves the dysregulation of circular RNAs. This study aimed to investigate the role of circular ubiquitin-specific peptidase 9 X-linked (circUSP9X) in AS cell models. Human umbilical vein endothelial cells (HUVECs) treated with oxidized low-density lipoprotein (ox-LDL) were used as cell models of AS. The expression of circUSP9X, miR-148b-3p, and Kruppel-like factor 5 (KLF5) messenger RNA was measured using quantitative polymerase chain reaction. Cell viability was assessed by Cell Counting Kit-8 assay. Lactate dehydrogenase leakage, malonaldehyde content, and superoxide dismutase activity were investigated using matched commercial kits. Cell apoptosis was detected using flow cytometry assay. The protein levels of apoptosis-related markers and KLF5 were detected by western blot. The release of proinflammatory factors was monitored by enzyme-linked immunosorbent assay. The predicted relationship between miR-148b-3p and circUSP9X or KLF5 was confirmed by dual-luciferase reporter assay or RNA immunoprecipitation assay. CircUSP9X was highly expressed in ox-LDL-treated HUVECs. CircUSP9X knockdown inhibited ox-LDL-induced lactate dehydrogenase leakage, apoptosis, inflammation, and oxidative stress in HUVECs. CircUSP9X directly bound to miR-148b-3p, and KLF5 was a target of miR-148b-3p. CircUSP9X could regulate KLF5 expression by competitively targeting miR-148b-3p. Rescue experiments indicated that circUSP9X knockdown inhibited ox-LDL-induced HUVEC injuries by enriching miR-148b-3p, and miR-148b-3p restoration alleviated ox-LDL-induced HUVEC injuries by degrading KLF5. In conclusion, circUSP9X knockdown relieved ox-LDL-triggered HUVEC injuries during AS progression partly by mediating the miR-148b-3p/KLF5 network.

Notoginsenoside R1 alleviates high glucose-induced inflammation and oxidative stress in HUVECs via upregulating miR-147a.

Endothelial dysfunction in atherosclerotic cardiovascular diseases has become one of the main characteristics in patients with diabetes mellitus, which is usually caused by abnormal inflammation and oxidative stress response. Presently, we focused on the role of Notoginsenoside R1 (NR1), a major component isolated from Panax notoginseng, in endothelial dysfunction caused by high glucose (HG). Human umbilical vein endothelial cells (HUVECs) were treated with HG and then dealt with NR1. Cell counting kit-8 assay and 5-bromo-2'-dexoyuridine assay were conducted to examine cell proliferation and viability. Flow cytometry was used to measure apoptosis. The angiogenesis of HUVECs was determined by tube formation assay. Moreover, the expressions of miR-147a, inflammatory cytokines (TNF-α, IL-6, and IL-10) and oxidative stress markers malondialdehyde, superoxide dismutase, and glutathione peroxidase were measured. The protein levels of MyD88/TRAF6/NF-κB axis, Bax, Bcl2, and Caspase3 were detected by Western blot. Furthermore, gain and loss of functional assays of miR-147a were performed to verify the role of miR-147a in NR1-mediated effects. Our data confirmed that NR1 (at 10-40 μM) reduces HG-induced HUVECs proliferation and viability inhibition, mitigates apoptosis, and enhances tube formation ability. Meanwhile, NR1 inhibited oxidative stress and inflammatory response and blocked the activation of the MyD88/TRAF6/NF-κB pathway induced by HG. In addition, NR1 promoted the expression of miR-147a, which targeted MyD88. Overexpression of miR-147a markedly inactivated MyD88/TRAF6/NF-κB pathway, while the miR-147a inhibitors reversed NR1-mediated protective effect in HG-induced HUVECs through activating MyD88/TRAF6/NF-κB pathway. In conclusion, NR1 relieves HG-induced endothelial cell injury by downregulating the MyD88/TRAF6/NF-κB pathway via upregulating miR-147a.

Knockdown of circ_0003204 alleviates oxidative low-density lipoprotein-induced human umbilical vein endothelial cells injury: Circulating RNAs could explain atherosclerosis disease progression.

Atherosclerosis (AS) is a serious cardiovascular disease. Circular RNAs (circRNAs) play an important role in the progression of many diseases, including AS. However, the role of circ_0003204 in AS is not clear. Oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) were used to construct an AS cell model in vitro. Cell viability was assessed using cell counting kit 8 (CCK8) assay. Flow cytometry and caspase-3 activity were used to measure cell apoptosis. The contents of inflammatory cytokines were measured using enzyme-linked immunosorbent assay (ELISA). Oxidative stress marker expression and cell injury marker activity were detected by their corresponding Assay Kits. Besides, the expression levels of circ_0003204, miR-330-5p, and toll-like receptor 4 (TLR4) were tested by real-time polymerase chain reaction (qPCR). The interaction between miR-330-5p and circ_0003204 or TLR4 was examined by dual-luciferase reporter assay and RNA pull-down assay. Western blot (WB) analysis was used to determine the levels of TLR4 protein and nuclear factor-kappa B (NF-κB) signaling pathway-related protein. Our data suggested that ox-LDL could suppress viability and promote apoptosis, inflammatory response, and oxidative stress in HUVECs. circ_0003204 was highly expressed in ox-LDL-induced HUVECs, and its silencing could inhibit ox-LDL-induced HUVECs injury. miR-330-5p could be sponged by circ_0003204, and its inhibitor could reverse the inhibition effect of silenced circ_0003204 on ox-LDL-induced HUVECs injury. Further, TLR4 could be targeted by miR-330-5p, and its overexpression could invert the suppression effect of miR-330-5p on ox-LDL-induced HUVECs injury. The activity of the NF-κB signaling pathway was regulated by the circ_0003204/miR-330-5p/TLR4 axis. Our results indicated that circ_0003204 silencing could alleviate ox-LDL-induced HUVECs injury, suggesting that circ_0003204 might be a novel target for AS treatment.

CLIC1 Inhibition Protects Against Cellular Senescence and Endothelial Dysfunction Via the Nrf2/HO-1 Pathway.

Chloride intracellular channel 1 (CLIC1) is a sensor of oxidative stress in endothelial cells (EC). However, the mechanism by which CLIC1 mediate the regulation of endothelial dysfunction has not been established. In this study, overexpressed CLIC1 impaired the ability of the vascular cells to resist oxidative damage and promoted cellular senescence. Besides, suppressed CLIC1 protected against cellular senescence and dysfunction in Human Umbilical Vein Endothelial Cells (HUVECs) through the Nrf2/HO-1 pathway. We also found that ROS-activated CLIC1-induced oxidative stress in HUVECs. Nrf2 nuclear translocation was inhibited by CLIC1 overexpression, but was enhanced by IAA94 (CLICs inhibitor) treatment or knockdown of CLIC1. The Nrf2/HO-1 pathway plays a critical role in the anti-oxidative effect of suppressing CLIC1. And inhibition of CLIC1 decreases oxidative stress injury by downregulating the levels of ROS, MDA, and the expression of EC effectors (ICAM1 and VCAM1) protein expression and promotes the activity of superoxide dismutase (SOD). The AMPK-mediated signaling pathway activates Nrf2 through Nrf2 phosphorylation and nuclear translocation, which is also regulated by CLIC1. Moreover, the activation of CLIC1 contributes to H2O2-induced mitochondrial dysfunction and activation of mitochondrial fission. Therefore, elucidation of the mechanisms by which CLIC1 is involved in these pivotal pathways may uncover its therapeutic potential in alleviating ECs oxidative stress and age-related cardiovascular disease development.

MiR-92a-3p promotes ox-LDL induced-apoptosis in HUVECs via targeting SIRT6 and activating MAPK signaling pathway.

Atherosclerosis could be induced by multiple factors, including hypertension, hyperlipidemia, and smoking, and its pathogenesis has not been fully elucidated. MicroRNAs have been shown to possess great anti-atherosclerotic potential, but the precise function of miR-92a-3p in atherosclerosis and its potential molecular mechanism have not been well clarified. Flow cytometry assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assay were performed to evaluate effects of oxidized low-density lipoprotein (ox-LDL) on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs), respectively. Malondialdehyde and superoxide dismutase levels in cell lysate were assessed with biochemical kits. The expression levels of miR-92a-3p and Sirtuin6 (SIRT6) in HUVECs exposed to ox-LDL were estimated by real-time quantitative polymerase chain reaction (RT-qPCR). In addition, the protein levels of SIRT6, c-Jun N-terminal kinase (JNK), phosphorylation JNK (p-JNK), p38 mitogen activated protein kinase (p38 MAPK), and phosphorylation p38 MAPK (p-p38 MAPK) were measured by western blot assays. The relationship between miR-92a-3p and SIRT6 was confirmed by dual-luciferase reporter assay. Ox-LDL induced apoptosis and oxidative stress in HUVECs in concentration- and time-dependent manners. Conversely, miR-92a-3p silencing inhibited apoptosis and SIRT6 expression in HUVECs. The overexpression of miR-92a-3p enhanced apoptosis and phosphorylation levels of JNK and p38 MAPK as well as inhibited proliferation in ox-LDL-induced HUVECs. In addition, SIRT6 was a target of miR-92a-3p. miR-92a-3p negatively regulated SIRT6 expression in ox-LDL-induced HUVECs to activate MAPK signaling pathway in vitro. In summary, miR-92a-3p promoted HUVECs apoptosis and suppressed proliferation in ox-LDL-induced HUVECs by targeting SIRT6 expression and activating MAPK signaling pathway.

Interference with lysophosphatidic acid receptor 5 ameliorates oxidized low-density lipoprotein-induced human umbilical vein endothelial cell injury by inactivating NOD-like receptor family, pyrin domain containing 3 inflammasome signaling

ABSTRACT Endothelial cell damage induced by oxidized low-density lipoprotein (ox-LDL) plays an important role in the pathogenesis of atherosclerosis (AS). We aimed to explore the effects of lysophosphatidic acid receptor 5 (LPAR5) on ox-LDL-induced damage of human umbilical vein endothelial cells (HUVECs). After HUVECs exposed to ox-LDL, LPAR5 expression was detected by RT-qPCR and western blotting. Then, LPAR5 was silenced and cell viability was determined with a CCK-8 assay. ELISA was employed to analyze the contents of inflammatory factors. The levels of oxidative stress markers were examined by kits. The expression of proteins related to endothelium function, including CD31, α-SMA, iNOS and eNOS, was evaluated with RT-qPCR and western blotting. Additionally, the effects of LPAR5 deletion on the NLRP3 inflammasome signaling in HUVECs under ox-LDL condition were assessed by determining NLRP3, caspase-1 and ASC expression. Afterward, NLRP3 agonist MSU was adopted for exploring the regulation of LPAR5 on NLRP3 inflammasome signaling in ox-LDL HUVECs injury. Results revealed that ox-LDL led to a significant upregulation in LPAR5 expression. NLRP3 knockdown enhanced cell viability, inhibited inflammation and oxidative stress in HUVECs after ox-LDL exposure. Besides, the expression of CD31 and eNOS was increased while that of α-SMA and iNOS was decreased after LPAR5 silencing. Moreover, interference with LPAR5 remarkably downregulated NLRP3, caspase-1 and ASC expression. Furthermore, MSU addition partially abrogated the inhibitory effects of LPAR5 deletion on the inflammation, oxidative stress and endothelium dysfunction of HUVECs. To conclude, we demonstrated that LPAR5 silencing alleviates ox-LDL-induced HUVECs injury by inhibiting NLRP3 inflammasome signaling.

Protective Effect of Simplicillium sp. Ethyl Acetate Extract against High Glucose-Induced Oxidative Stress in HUVECs.

This study aimed at investigating the cytoprotective effect of an ethyl acetate extract of insect fungi against high glucose- (HG-) induced oxidative damage in human umbilical vein endothelial cells (HUVECs). An insect fungus strain termed CH180672 (CH) was found for protecting HUVECs from HG-induced damage. In this study, CH was identified as Simplicillium sp. based on a phylogenetic analysis of ITS‐rDNA sequences. Ethyl acetate extract (EtOAc) of this strain (CH) was subjected to the following experiments. Cell viability was examined with the MTT method. To evaluate the protection of CH, intracellular reactive oxygen species (ROS), malondialdehyde (MDA) levels, and the activities of antioxidant enzymes were measured and the expression of oxidation-associated proteins was assessed. In the current study, it has been found that CH can increase the survival rate of HUVECs induced by HG. Additionally, we found that HG-induced nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) signal decreased and increased the intracellular ROS and MDA generation in HUVECs. However, CH treatment strongly promoted the translocation of Nrf2 and its transregulation on HO-1 and ultimately inhibited the high level of ROS and MDA induced by HG. The regulatory ability of CH was similar to Nrf2 agonist bardoxolone, while the effect was abolished by ML385, suggesting that Nrf2 mediated the inhibition of CH on HG-induced oxidative stress in HUVECs. Taken together, CH can improve HG-induced oxidative damage of HUVECs, and its mechanism may be related to the regulation of the Nrf2/HO-1 pathway.

LPS promotes the progression of sepsis by activation of lncRNA HULC/miR-204-5p/TRPM7 network in HUVECs.

Sepsis is a systemic inflammatory response syndrome caused by infection. Lipopolysaccharide (LPS) has been reported to induce inflammatory responses, and long non-coding RNA highly up-regulated in liver cancer (HULC) expression was associated with the progression of sepsis. But the role and underlying mechanism of HULC in LPS-induced sepsis remain unclear. Cell viability and apoptosis were measured by methyl thiazolyl tetrazolium (MTT) and flow cytometry assays, respectively. The levels of apoptosis-related proteins, inflammatory cytokines and transient receptor potential melastatin7 (TRPM7) were detected by western blot. Reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA) levels were detected by dichloro-dihydro-fluorescein diacetate (DCFH-DA) method using commercial kit. HULC, microRNA-204-5p (miR-204-5p) and TRPM7 expressions in serum of sepsis patients and human umbilical vein endothelial cells (HUVECs) were examined by quantitative real-time polymerase chain reaction (qRT-PCR). Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to confirm the interaction between HULC and miR-204-5p, miR-204-5p and TRPM7. LPS stimulation restrained cell viability and facilitated apoptosis, inflammatory injury and oxidative stress in HUVECs. HULC and TRPM7 were increased and accompanied with decreased miR-204-5p expression in serum of sepsis patients. A significant negative correlation between miR-204-5p and HULC or TRPM7 was observed, and there was a positive relationship between expressions of HULC and TRPM7. Importantly, LPS inhibited the cell viability and induced apoptosis, inflammatory injury and oxidative stress of HUVECs by up-regulating the expressions of HULC and TRPM7, and down-modulating miR-204-5p expression. Mechanically, HULC positively regulated TRPM7 expression by sponging miR-204-5p in HUVECs. LPS impaired cell viability, and promoted cell apoptosis, inflammatory response and oxidative stress in HUVECs by regulating HULC/miR-204-5p/TRPM7 axis.

Proteomic analysis of oxidative stress response in human umbilical vein endothelial cells (HUVECs): role of heme oxygenase 1 (HMOX1) in hypoxanthine-induced oxidative stress in HUVECs.

BackgroundErectile dysfunction (ED) is a well-known complication of diabetes, affecting up to 75% of diabetic men. Although the etiology of diabetic ED is multifactorial, endothelial dysfunction is considered to be a pillar of its pathophysiology. Endothelial dysfunction is caused by the harmful effects of high glucose levels and increased oxidative stress on the endothelial cells that comprise the vascular endothelium. The aim of this study was to identify the proteomic changes caused by high glucose-induced oxidative stress and explore the role of heme oxygenase 1 (HMOX1) in it.MethodsThe cellular proteomic response to hypoxanthine-induced oxidative stress in human umbilical vein endothelial cells (HUVECs) was analyzed by isobaric tags for relative and absolute quantitation (iTRAQ) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins (DEPs) were analyzed through Network and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Further validation assays was performed to validate the role of HMOX1.ResultsThe results showed that 66 and 76 DEPs were markedly upregulated and downregulated, respectively, for HUVECs oxidative stress. Among these proteins, we verified eight dysregulated genes by quantitative reverse transcription PCR, including nucleolin (NCL), X-ray repair cross-complementing protein 6 (XRCC6), ubiquinol-cytochrome C reductase binding protein (UQCRB), non-POU domain containing octamer binding (NONO), heme oxygenase 1 (HMOX1), nucleobindin 1 (NUCB1), DEK, and chromatin target of prmt1 (CHTOP). Further, using overexpression and genetic knockdown approaches, we found that HMOX1 was critical for the oxidative stress response in HUVECs.ConclusionsWe found that HMOX1 was closely related to the oxidative stress response induced by hypoxanthine. To the best of our knowledge, this study is the first overview of the responses of HUVECs to oxidative stress. The findings will contribute to analyses of the detailed molecular mechanisms involved in the pathogenesis of endothelial dysfunction and related molecular mechanisms in ED patients.

LncRNA XIST knockdown ameliorates oxidative low-density lipoprotein-induced endothelial cells injury by targeting miR-204-5p/TLR4

Oxidative low-density lipoprotein (ox-LDL)-induced endothelial cell injury is a key contributor to atherosclerosis development. However, the role and mechanism of long noncoding RNA X-inactive specific transcript (XIST) in atherosclerosis remain largely unknown. The ox-LDL-induced human umbilical vein endothelial cells (HUVECs) injury was analyzed by cell viability, apoptosis, inflammatory cytokines secretion and oxidative stress. The expression levels of XIST, microRNA-204-5p (miR-204-5p) and toll-like receptor 4 (TLR4) were detected by quantitative real-time polymerase chain reaction and western blot, respectively. The target interaction between miR-204-5p and XIST or TLR4 was explored by bioinformatics analysis, luciferase assay and RNA immunoprecipitation. The expression of XIST was enhanced in ox-LDL-treated HUVECs. Knockdown of XIST attenuated ox-LDL-induced viability inhibition, apoptosis production, inflammatory response and oxidative stress in HUVECs. XIST was validated as a sponge of miR-204-5p and TLR4 acted as a target of miR-204-5p. Knockdown of miR-204-5p reversed silence of XISTmediated suppressive role in ox-LDL-induced injury. TLR4 alleviated miR-204-5p-mediated inhibitive effect on ox-LDL-induced injury. Moreover, XIST could regulate TLR4 expression by sponging miR-204-5p. In conclusion, silence of XIST displayed a protective role in ox-LDL-induced injury in HUVECs by regulating miR-204-5p/TLR4 axis, providing a novel mechanism for understanding the pathogenesis of atherosclerosis.

Endosulfan promotes cell migration via PTP4A3-mediated signaling pathways in HUVECs.

Endosulfan is a persistent organic pollutant and can cause endothelial dysfunction, closely related to cardiovascular diseases. Endothelial cell migration plays a critical role in atherosclerosis and angiogenesis. This study was aimed to investigate the effect of environmentally relevant doses of endosulfan and underlying molecular mechanism on endothelial cell migration. Human umbilical vein endothelial cells (HUVECs) were treated with DMSO (control) or endosulfan (0.1, 1, 10 and 20μM) in the presence or absence of inhibitors. Wound healing and Transwell assay were employed to explore the effect of endosulfan on endothelial cell migration. The expression of genes or proteins was assayed by real-time PCR or immunoblotting. The results showed that endosulfan at relative low concentration (0.1, 1, 10 and 20μM) increased cell migration ability horizontally and vertically at 12h after exposure. In line with this cellular effect, Protein-tyrosine Phosphatase 4A3 (PTP4A3) expression was significantly increased in endosulfan-exposed endothelial cells. Specific inhibitor of PTP4A3 significantly inhibited 20μM endosulfan-induced cell migration, the expression and phosphorylation of Src and phosphorylation of focal adhesion kinase (FAK). Exposure to endosulfan resulted in activation of various signaling pathways including phosphoinositide 3-kinase (PI3K)/AKT, mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB), which were suppressed by PTP4A3 inhibitor or specific inhibitor for each signaling pathway. Exposure to endosulfan significantly reduced nitric oxide production and caused oxidative stress in HUVECs. These findings suggest that endosulfan promoted cell migration through PTP4A3-mediated various signaling pathways in endothelial cells.