Human Umbilical Vein Endothelial Cells Injury Research Articles

METTL3-mediated NDUFB5 m6A modification promotes cell migration and mitochondrial respiration to promote the wound healing of diabetic foot ulcer

BackgroundDiabetic foot ulcer (DFU) is the most devastating complication of diabetes mellitus (DM) and plays a major role in disability and death in DM patients. NADH: ubiquinone oxidoreductase subunit B5 (NDUFB5) plays an important role in maintaining mitochondrial respiration, but whether it is involved in regulating the progression of advanced glycation end products (AGEs)-mediated DFU is still unclear.MethodsFirstly, the role of AGEs on cell viability, migration, and mitochondrial respiration in human umbilical vein endothelial cells (HUVECs) was explored in vitro. Next, NDUFB5 expression was detected in human samples and AGEs-treated HUVECs, and NDUFB5’s effect on AGEs-induced HUVECs injury and skin wound in diabetic mice was further clarified. In addition, the role of m6A modification mediated by methyltransferase-like 3 (METTL3) in regulating NDUFB5 expression and AGEs-induced HUVECs injury was investigated.ResultsNDUFB5 promoted cell viability, migration, and mitochondrial respiration in AGEs-treated HUVECs, whereas mitochondrial fusion promoter M1 facilitated cell viability, migration, and mitochondrial oxiadative respiration in NDUFB5 knockdown HUVECs. Meanwhile, NDUFB5 promotes skin wound healing in diabetic mice. Besides, METTL3-mediated m6A modification and insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) enhanced NDUFB5 expression in HUVECs. Furthermore, METTL3 promoted cell viability, migration, and mitochondrial respiration in AGEs-treated HUVECs by increasing NDUFB5.ConclusionMETTL3-mediated NDUFB5 m6A modification inhibits AGEs-induced cell injury in HUVECs. METTL3 and NDUFB5 might serve as potential targets for DFU therapy in the future.

METTL3-mediated NDUFB5 m6A modification promotes cell migration and mitochondrial respiration to promote the wound healing of diabetic foot ulcer

BackgroundDiabetic foot ulcer (DFU) is the most devastating complication of diabetes mellitus (DM) and plays a major role in disability and death in DM patients. NADH: ubiquinone oxidoreductase subunit B5 (NDUFB5) plays an important role in maintaining mitochondrial respiration, but whether it is involved in regulating the progression of advanced glycation end products (AGEs)-mediated DFU is still unclear.MethodsFirstly, the role of AGEs on cell viability, migration, and mitochondrial respiration in human umbilical vein endothelial cells (HUVECs) was explored in vitro. Next, NDUFB5 expression was detected in human samples and AGEs-treated HUVECs, and NDUFB5’s effect on AGEs-induced HUVECs injury and skin wound in diabetic mice was further clarified. In addition, the role of m6A modification mediated by methyltransferase-like 3 (METTL3) in regulating NDUFB5 expression and AGEs-induced HUVECs injury was investigated.ResultsNDUFB5 promoted cell viability, migration, and mitochondrial respiration in AGEs-treated HUVECs, whereas mitochondrial fusion promoter M1 facilitated cell viability, migration, and mitochondrial oxiadative respiration in NDUFB5 knockdown HUVECs. Meanwhile, NDUFB5 promotes skin wound healing in diabetic mice. Besides, METTL3-mediated m6A modification and insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) enhanced NDUFB5 expression in HUVECs. Furthermore, METTL3 promoted cell viability, migration, and mitochondrial respiration in AGEs-treated HUVECs by increasing NDUFB5.ConclusionMETTL3-mediated NDUFB5 m6A modification inhibits AGEs-induced cell injury in HUVECs. METTL3 and NDUFB5 might serve as potential targets for DFU therapy in the future.

Circ_0005699 Expedites ox-LDL-Triggered Endothelial Cell Injury via Targeting miR-384/ASPH Axis.

Atherosclerosis (AS) is an inflammatory disease with multiple causes. Multiple circular RNAs (circRNAs) are known to be involved in the pathogenesis of AS. To explore the function and mechanism of circ_0005699 in oxidative low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) injury. Ox-LDL treatment restrained HUVECs viability, cell proliferation, and angiogenesis ability, and accelerated HUVECs apoptosis, inflammatory response, and oxidative stress. Circ_0005699 was up-regulated in the serum samples of AS patients and ox-LDL-induced HUVECs. Interference of circ_0005699 effectively rescued ox-LDL-induced injury in HUVECs. Additionally, miR-384 could bind to circ_0005699, and miR-384 depletion inverted the effects of circ_0005699 deficiency on ox-LDL-mediated HUVEC injury. Moreover, ASPH was a direct target of miR-384, and the enforced expression of ASPH overturned miR-384-induced effects on ox-LDL-induced HUVECs. Importantly, circ_0005699 regulated ASPH expression via sponging miR-384. Interference of circ_0005699 protected against ox-LDL-induced injury in HUVECs at least partly by regulating ASPH expression via acting as a miR-384 sponge.

Ciliary neurotrophic factor promotes the development of homocysteine-induced vascular endothelial injury through inflammation mediated by the JAK2/STAT3 signaling pathway

Elevated homocysteine (Hcy) levels have been recognized as significant risk factor for cardiovascular and cerebrovascular diseases, closely related to endothelial injury. While expression of Ciliary Neurotrophic Factor (CNTF) significantly increases during Hcy-induced vascular endothelial cell injury, the precise molecular pathways through which CNTF operates remain to be clarified. To induce vascular endothelial cell injury, human umbilical vein endothelial cells (HUVECs) were treated with Hcy. Cell viability and apoptosis in HUVECs were assessed using the CCK-8 assay and flow cytometry. Western blot analysis determined the expression levels of the JAK2-STAT3 pathway, inflammation-related factors (IL-1β, NLRP3, ICAM-1, VCAM-1), and apoptosis-related factors (cleaved Caspase-3 and Bax). Immunofluorescence staining and western blotting were employed to examine CD31 and α-SMA expression. Knockdown of CNTF was achieved using lentiviral interference, and its effects on inflammation and cell injury were evaluated. Chromatin immunoprecipitation (ChIP) and dual luciferase reporter analysis were conducted to investigate the interaction between the MAFK and CNTF promoters. Our results indicated that Hcy induced high expression of CNTF and activated the JAK2-STAT3 signaling pathway, thereby upregulating factors associated with inflammation and cell apoptosis. Inhibiting CNTF alleviated Hcy-induced inflammation and cell injury. MAFK was identified as a transcription factor promoting CNTF transcription, and its overexpression exacerbated inflammation and cell injury in Hcy-treated HUVECs through the CNTF-JAK2-STAT3 axis, which could be reversed by knocking down CNTF. Activation of MAFK leads to CNTF upregulation, which activates the JAK2-STAT3 signaling pathway, regulating inflammation and inducing injury in Hcy-exposed vascular endothelial cells. Targeting CNTF or its upstream regulator MAFK may represent potential therapeutic strategies for mitigating endothelial dysfunction associated with hyperhomocysteinemia and cardiovascular diseases.

PCSK9 induces endothelial cell autophagy by regulating the PI3K/ATK pathway in atherosclerotic coronary heart disease.

Atherosclerosis is a chronic inflammatory disease of the arteries, and its pathogenesis is related to endothelial dysfunction. It has been found that the protein convertase subtilin/kexin9 type (PCSK9) plays an important role in AS, but its specific mechanism is still unclear. In this study, we first cultured human umbilical vein endothelial cells (HUVECs) with 50 or 100μg/ml oxidized low-density lipoprotein (ox-LDL) for 24 hours to establish a coronary atherosclerosis cell model. The results showed that ox-LDL induced HUVEC injury and autophagy and upregulated PCSK9 protein expression in HUVECs in a concentration-dependent manner. Silencing PCSK9 expression with siRNA inhibited ox-LDL-induced HUVEC endothelial dysfunction, inhibited the release of inflammatory factors, promoted HUVEC proliferation and inhibited apoptosis. In addition, ox-LDL increased the expression of LC3B-I and LC3B-II and decreased the expression of p62. However, these processes are reversed by sh-PCSK9. In addition, sh-PCSK9 can inhibit PI3K, AKT and mTOR phosphorylation and promote autophagy. Taken together, our research shows that silencing PCSK9 inhibits the PI3K/ATK/mTOR pathway to activate ox-LDL-induced autophagy in vascular endothelial cells, alleviating endothelial cell injury and inflammation.

Up-regulation of Trim28 in pregnancy-induced hypertension is involved in the injury of human umbilical vein endothelial cells through the p38 signaling pathway.

The present study is to analyze the regulation and potential molecular mechanism of Trim28 on vascular endothelial injury induced by pregnancy-induced hypertension (PIH). Trim28 mRNA in placental tissues and peripheral blood from PIH patients were determined by quantitative real-time polymerase chain reaction. The serum from PIH was used to stimulate human umbilical vein endothelial cells (HUVECs). After silencing Trim28 in HUVECs, we used CCK-8 assay, Transwell assay and flow cytometry to investigate proliferation, migration and apoptosis. Western blotting was used to measure Trim28 protein level and p38 phosphorylation level. After addition of p38 inhibitor, the proliferation, migration and apoptosis of HUVECs with silenced Trim28 were studied again. Trim28 expression in placental tissues and peripheral blood from PIH patients is elevated, and serum from these patients can up-regulate the expression of Trim28 in HUVECs in vitro. Trim28 silencing significantly inhibits the proliferation and migration of HUVECs by affecting the cell cycle. Down-regulation of Trim28 expression promotes the apoptosis of HUVECs. Trim28 regulates the biological function of HUVECs by affecting the activity of the p38 signaling pathway. The present study demonstrates that Trim28 is up-regulated in peripheral blood of patients with PIH and participates in HUVECs injury through the p38 signaling pathway.

Uvaol alleviates oxidative stress induced human umbilical vein endothelial cell injury by suppressing mitogen-activated protein kinase signaling pathway.

Deep venous thrombosis (DVT) is a potentially life-threatening disorder with high morbidity. Uvaol is a natural pentacyclic triterpene possessing multiple pharmacological activities. Nevertheless, the role of uvaol in DVT is unclarified. Human umbilical vein endothelial cells (HUVECs) were treated with hydrogen peroxide (H 2 O 2 ) to mimic DVT in vitro . CCK-8 assay and flow cytometry were utilized for measuring cell viability and apoptosis, respectively. Levels of the cell injury marker, thrombosis-associated factors, inflammatory cytokines, and oxidative stress-related markers were examined by commercial assay kits. Western blotting was used for evaluating the expression of mitogen-activated protein kinase (MAPK) signaling-associated proteins. Uvaol treatment attenuated H 2 O 2 -induced HUVEC apoptosis and injury. Uvaol reduced the expression of pro-thrombotic factors and inflammatory cytokines and attenuated oxidative stress in H 2 O 2 -stimulated HUVECs. Uvaol inhibited MAPK signaling pathway in H 2 O 2 -stimulated HUVECs. Activating MAPK signaling reversed uvaol-mediated protective effects on H 2 O 2 -treated HUVECs. Uvaol treatment alleviates H 2 O 2 -induced HUVEC injury, apoptosis, and oxidative stress by inactivating MAPK signaling.

MiR-328-3p targets TLR2 to ameliorate oxygen-glucose deprivation injury and neutrophil extracellular trap formation in HUVECs via inhibition of the NF-κB signaling pathway.

Endothelial cell injury is one of the important pathogenic mechanisms in thrombotic diseases, and also neutrophils are involved. MicroRNAs (miRNAs) have been demonstrated to act as essential players in endothelial cell injury, but the potential molecular processes are unknown. In this study, we used cellular tests to ascertain the protective effect of miR-328-3p on human umbilical vein endothelial cells (HUVECs) treated with oxygen-glucose deprivation (OGD). In our study, an OGD-induced HUVECs model was established, and we constructed lentiviral vectors to establish stable HUVECs cell lines. miR-328-3p and Toll-like receptor 2 (TLR2) interacted, as demonstrated by the dual luciferase reporter assay. We used the CCK8, LDH release, and EdU assays to evaluate the proliferative capacity of each group of cells. To investigate the expression of TLR2, p-P65 NF-κB, P65 NF-κB, NLRP3, IL-1β, and IL-18, we employed Western blot and ELISA. Following OGD, each group's cell supernatants were gathered and co-cultured with neutrophils. An immunofluorescence assay and Transwell assay have been performed to determine whether miR-328-3p/TLR2 interferes with neutrophil migration and neutrophil extracellular traps (NETs) formation. In OGD-treated HUVECs, the expression of miR-328-3p is downregulated. miR-328-3p directly targets TLR2, inhibits the NF-κB signaling pathway, and reverses the proliferative capacity of OGD-treated HUVECs, while inhibiting neutrophil migration and neutrophil extracellular trap formation. miR-328-3p inhibits the NF-κB signaling pathway in OGD-treated HUVECs while inhibiting neutrophil migration and NETs formation, and ameliorating endothelial cell injury, which provides new ideas for the pathogenesis of thrombotic diseases.

Molecular Mechanism of SOX18 in Lipopolysaccharide-Induced Injury of Human Umbilical Vein Endothelial Cells.

Endothelial dysfunction is associated with the progression of sepsis. This study sought to probe the molecular route of sex-determining region on the Y chromosome-box transcription factor 18 (SOX18) in sepsis-associated endothelial injury. Human umbilical vein endothelial cells (HUVECs) were treated with lipopolysaccharide (LPS) to establish the sepsis cell model. Cell viability, lactate dehydrogenase (LDH) release, oxidative stress (reactive oxygen species/malondialdehyde/superoxide dismutase), and inflammation (interleukin-1β/tumor necrosis factor-α/interleukin-6) were evaluated by cell counting kit-8 assay and relevant assay kits. The expression levels of SOX18, microRNA (miR)-204-5p, and cadherin-2 (CDH2) in cells were determined by real-time quantitative polymerase chain reaction and Western blot assay. The interaction of SOX18, miR-204-5p, and CDH2 was analyzed by chromatin immunoprecipitation and dual-luciferase assay. LPS induced HUVECs injury and downregulation of SOX18. SOX18 overexpression increased cell viability, while decreased LDH activity, oxidative stress, and inflammation. SOX18 bound to the miR-204-5p promoter to promote miR-204-5p expression, and further repressed CDH2 expression. miR-204-5p knockdown and CDH2 overexpression abrogated the protective role of SOX18 in HUVECs injury. Overall, SOX18 alleviated LPS-induced injury of HUVECs by promoting miR-204-5p and repressing CDH2, suggesting it as a potential target for sepsis treatment.

Asiaticoside Prevents Oxidative Stress and Apoptosis in Endothelial Cells by Activating ROS-dependent p53/Bcl-2/Caspase-3 Signaling Pathway.

Asiaticoside (AC) is a triterpenoid saponin found in Centella asiatica (L.) urban extract that has a wide range of pharmacological properties. Our previous study demonstrated that AC could promote angiogenesis in diabetic wounds, but the specific mechanisms remain unknown. This study aimed to examine the effectiveness and mechanism of AC on human umbilical vein endothelial cells (HUVECs) exposed to tert-butyl hydroperoxide (t-BHP) toxicity. Senescence was confirmed using senescence-associated betagalactosidase (SA-β-gal) activity and expression of the cell cycle phase markers p16 and p21. The levels of SOD, NO, MDA, GSH-Px, and ROS were tested. Furthermore, several cell death-related genes and proteins (p53, Bax, Bcl-2 and Caspase-3) were assessed with RT-qPCR and Western blotting. AC significantly reduced SA-β-gal activity, with both the suppression of cellcycle inhibitors p16 and p21. We also found that the induced oxidative stress and apoptosis caused by t-BHP treatment resulted in the decrease of antioxidant enzymes activities, the surge of ROS and MDA, the up-regulation of p53, Bax and caspase-3, and the decrease of SOD, NO, GSH-Px and Bcl-2. These biochemical changes were all reversed by treatment with varying doses of AC. AC alleviates t-BHP-induced oxidative injury and apoptosis in HUVECs through the ROS-dependent p53/Bcl-2/Caspase-3 signaling pathway. It may be a potential antioxidant applied in metabolic disorders and pharmaceutical products.

Anti-hypertensive and composition as well as pharmacokinetics and tissues distribution of active ingredients from Alpinia zerumbet

Alpinia zerumbet is a food flavor additive and a traditional medicine herb around the world. Several studies have reported that A. zerumbet has excellent effects on a variety of cardiovascular diseases, but its potential hypertensive applications, and pharmacokinetic features of main active substances have not been fully investigated. The mechanism of anti-hypertension with ethyl acetate extracts of A. zerumbet fruits (AZEAE) was evaluated by L-NNA-induced hypertensive rats and L-NAME-injured human umbilical vein endothelial cells (HUVECs). Blood pressure, echocardiographic cardiac index and H&E staining were used to preliminary evaluate the antihypertensive effect of AZEAE, the levels of TNF-α, IL-6, and IL-1β were evaluated by ELISA, and the proteins expression of IL-1β, IL-18, AGTR1, VCAM, iNOS, EDN1 and eNOS were also evaluated. In addition, isolation, identification, and activity screening of bioactive compounds were carried ou. Next, pharmacokinetics and tissues distribution of dihydro-5,6-dehydrokavain (DDK) in vivo were measured, and preliminary absorption mechanism was conducted with Caco-2 cell monolayers. AZEAE remarkably enhanced the state of hypertensive rats. Twelve compounds were isolated and identified, and five compounds were isolated from this plant for the first time. The isolated compounds also exhibited good resistance against injury of HUVECs. Moreover, pharmacokinetics and Caco-2 cell monolayers demonstrated AZEAE had better absorption capacity than DDK, and DDK exhibited differences in tissues distribution and gender difference. This study was the first to assess the potential hypertensive applications of A. zerumbet in vivo and vitro, and the first direct and concise study of the in vivo behavior of DDK and AZEAE.

Danshenxinkun B protects human umbilical vein endothelial cells against ox-LDL-induced injury by inhibiting pyroptosis and the NF-κB/NLRP3 pathway

To investigate the protective effect of Danshenxinkun B against oxidized low-density lipoprotein (ox-LDL)- induced human umbilical vein endothelial cell (HUVEC) injury and explore the underlying mechanism. HUVECs cultured in the presence of 10% fetal bovine serum were treated with ox-LDL (100 μg/mL), ox-LDL+0.1% dimethyl sulfoxide (DMSO), or ox-LDL+Danshenxinkun B (100 ng/mL, dissolved in DMSO) for 24 h. The changes in lactate dehydrogenase (LDH) release was detected, and qRT-PCR was used to detect the mRNA expressions of nuclear factor-κB1 (NF-κB1), nucleotide binding oligomerization domain-like receptor family pyrin domain protein 3 (NLRP3), gasdermin D (GSDMD) and interleukin- 1β (IL-1β). The protein expressions of NF-κB1, NLRP3, caspase-1, IL-1β and GSDMD-N were detected with Western blotting. Immunofluorescence assay was performed to examine the changes in GSDMD expression in the cells. Compared with the normal control cells, the cells treated with ox-LDL alone or in combination with DMSO exhibited significantly increased LDH release, mRNA expressions of NF-κB1, NLRP3, GSDMD, and IL- 1β and the protein levels of NF-κB1, NLRP3, IL- 1β, GSDMD-N and caspase-1 (P<0.01), which were all significantly lowered by treatment with Danshenxinkun B (P<0.05 or 0.01). Danshenxinkun B treatment significantly inhibited GSDMD expression on the cell membrane and restricted its entry into the cell nucleus. Danshenxinkun B alleviates ox-LDL-induced HUVEC injury possibly by suppressing pyroptosis mediated by NLRP3 inflammatory bodies and inhibiting the NF-κB/NLRP3 signaling pathway.

Inducible nitric oxide synthase activity mediates TNF-α-induced endothelial cell dysfunction.

Inducible nitric oxide synthase (iNOS) and vascular endothelial dysfunction have been implicated in the development and progression of atherosclerosis. This study aimed to elucidate the role of iNOS in vascular endothelial dysfunction. Ultrahigh performance liquid chromatography-quadrupole time-of-flight mass spectrometry combined with multivariate data analysis was used to characterize the metabolic changes in human umbilical vein endothelial cells (HUVECs) in response to different treatment conditions. In addition, molecular biology techniques were employed to explain the molecular mechanisms underlying the role of iNOS in vascular endothelial dysfunction. Tumor necrosis factor-α (TNF-α) enhances the expression of iNOS, TXNIP, and the level of reactive oxygen species (ROS) facilitates the entry of nuclear factor-κB (NF-κB) into the nucleus and promotes injury in HUVECs. iNOS deficiency reversed the TNF-α-mediated pathological changes in HUVECs. Moreover, TNF-α increased the expression of tumor necrosis factor receptor-2 (TNFR-2) and the levels of p-IκBα and IL-6 proteins and CD31, ICAM-1, and VCAM-1 protein expression, which was significantly reduced in HUVECs with iNOS deficiency. In addition, treating HUVECs in the absence or presence of TNF-α or iNOS, respectively, enabled the identification of putative endogenous biomarkers associated with endothelial dysfunction. These biomarkers were involved in critical metabolic pathways, including glycosylphosphatidylinositol-anchor biosynthesis, amino acid metabolism, sphingolipid metabolism, and fatty acid metabolism. iNOS deficiency during vascular endothelial dysfunction may affect the expression of TNFR-2, vascular adhesion factors, and the level of ROS via cellular metabolic changes, thereby attenuating vascular endothelial dysfunction.NEW & NOTEWORTHY Inducible nitric oxide synthase (iNOS) deficiency during vascular endothelial dysfunction may affect the expression of tumor necrosis factor receptor-2 and vascular adhesion factors via cellular metabolic changes, thereby attenuating vascular endothelial dysfunction.

Gastrodin protects endothelial cells against high glucose-induced injury through up-regulation of PPARβ and alleviation of nitrative stress.

In diabetes mellitus (DM), high glucose can result in endothelial cell injury, and then lead to diabetic vascular complications. Gastrodin, as the mainly components of Chinese traditional herb Tianma (Gastrodia elata Bl.), has been widely used for cardiovascular diseases. However, the known of the effect of gastrodin on endothelial cell injury is still limited. In this study, we aimed to investigate the effect and possible mechanism of gastrodin on high glucose-injured human umbilical vein endothelial cells (HUVEC). High glucose (30mmol/L) treatment caused HUVEC injury. After gastrodin (0.1, 1, 10μmol/L) treatment, compared with the high glucose group, the cell proliferation ability increased in a dose-dependent manner. Meanwhile, gastrodin (10μmol/L) up-regulated the mRNA and protein expressions of PPARβ and eNOS, decreased the expressions of iNOS, also reduced the protein expression of 3-nitrotyrosine, and lowed the level of ONOO-, increased NO content. Both the PPARβ antagonist GSK0660 (1μmol/L) and the eNOS inhibitor L-NAME (10μmol/L) were able to block the above effects of gastrodin. In conclusion, gastrodin protectes vascular endothelial cells from high glucose injury, which may be, at least partly, mediated by up-regulating the expression of PPARβ and negatively regulating nitrative stress.

Circ_0033596 depletion ameliorates oxidized low-density lipoprotein-induced human umbilical vein endothelial cell damage.

Previous data have shown that circ_0033596 is involved in the pathogenesis of atherosclerosis (AS). The study aims to reveal the detailed mechanism of circ_0033596 in AS. Human umbilical vein endothelial cells (HUVECs) were treated with oxidized low-density lipoprotein (ox-LDL) to establish an AS cell model. Quantitative real-time polymerase chain reaction and western blot were implemented to detect the expression of circ_0033596, miR-637, growth factor receptor bound protein2 (GRB2), BCL2-associated x protein (Bax) and B-cell lymphoma-2 (Bcl-2). Cell viability, proliferation, apoptosis and tube formation were investigated by cell counting kit-8, EdU assay, flow cytometry and tube formation assay, respectively. The production of interleukin (IL-6) and tumor necrosis factor-α (TNF-α) was evaluated by enzyme-linked immunosorbent assay. Oxidative stress was evaluated by lipid peroxidation malondialdehyde assay kit and superoxide dismutase activity assay kit. Dual-luciferase reporter assay, RNA pull-down assay and RIP assay were performed to identify the associations among circ_0033596, miR-637 and GRB2. The expression of circ_0033596 and GRB2 was significantly increased, while miR-637 was decreased in the blood of AS patients and ox-LDL-induced HUVECs compared with controls. Ox-LDL treatment inhibited HUVEC viability, proliferation and angiogenic ability and induced cell apoptosis, inflammation and oxidative stress, while these effects were attenuated after circ_0033596 knockdown. Circ_0033596 interacted with miR-637 and regulated ox-LDL-induced HUVEC damage by targeting miR-637. In addition, GRB2, a target gene of miR-637, participated in ox-LDL-induced HUVEC injury by combining with miR-637. Importantly, circ_0033596 activated GRB2 by interacting with miR-637. Circ_0033596 depletion protected against ox-LDL-induced HUVEC injury by miR-637/GRB2 pathway, providing a therapeutic target for AS.

Circular RNA circ_0029589 promotes ox-LDL-induced endothelial cell injury through regulating RAB22A by serving as a sponge of miR-1197.

Dysfunction of endothelial cells is now considered a vital contributor to the pathogenesis of atherosclerosis (AS). Moreover, circular RNA (circRNA) circ_0029589 has been found to be involved in the regulation of oxidized low-density lipoprotein (ox-LDL)-induced endothelial cell damage. Nevertheless, its molecular mechanism in ox-LDL-triggered endothelial cell injury is poorly defined. Human umbilical vein endothelial cells (HUVECs) treated with ox-LDL were applied as cell models of AS. Circ_0029589, microRNA-1197 (miR-1197), and Ras-related protein Rab-22A (RAB22A) expression were detected using real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation, apoptosis, angiogenesis, and invasion were detected using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, tube formation, and transwell assays. Western blot analysis of Cleaved-caspase-3, B-cell lymphoma-2 related X protein (Bax), and RAB22A. IL-6, IL-1β, and Tumor necrosis factor α (TNF-α) levels were gauged using ELISA kits. Superoxide Dismutase (SOD) activity and Malondiahyde (MDA) level were assessed using special kits. Bioinformatics software predicted the binding between miR-1197 and circ_0029589 or RAB22A, which was proved using dual-luciferase reporter and RNA pull-down assays. Circ_0029589 and RAB22A expression were strengthened, and miR-1197 was reduced in ox-LDL-treated HUVECs. Importantly, circ_0029589 silencing ameliorated ox-LDL-triggered HUVEC damage via promoting cell proliferation, tube formation ability, invasion, and repressing cell apoptosis, inflammation, and oxidative stress. Mechanical analysis suggested that circ_0029589 might affect RAB22A content through sponging miR-1197. Circ_0090231 might protect against ox-LDL-mediated HUVEC injury via the miR-1197/RAB22A axis, which provides a therapeutic strategy for endothelial cell damage of AS.

Echinacoside ameliorates 5-fluorouracil-induced endothelial injury and senescence through SIRT1 activation

Echinacoside (ECH) is a natural bioactive component with antioxidant, anti-inflammatory, anti-apoptosis, and anti-tumor properties. In the current study, we explore the ECH-mediated protective effect and underlying mechanism of 5-fluorouracil (5-FU)-induced endothelial injury and senescence in the Human umbilical vein endothelial cells (HUVECs). In HUVECs, Cell viability, Apoptosis and Senescence assays evaluated 5-fluorouracil-induced endothelial injury and senescence. Protein expressions were assessed using RT-qPCR and Western blotting. Our results showed that 5-FU-induced endothelial injury and endothelial cell senescence could be improved when treated with ECH in HUVECs. ECH treatment potentially attenuated oxidative stress and ROS production in HUVECs. In addition, the effect of ECH on autophagy markedly reduced the percentage of HUVECs with LC3-II dots and suppressed the Beclin-1 and ATG7 mRNA expression but enhanced the p62 mRNA expression. Besides, ECH treatment significantly increased migrated cells and suppressed the adhesion of THP-1 monocytes in HUVECs. Furthermore, ECH treatment activated the SIRT1 pathway, and its related proteins (SIRT1, p-AMPK and eNOS) expression increased. Nicotinamide (NAM), an inhibitor of SIRT1, significantly attenuated the ECH-induced decrease in the apoptotic rate, increased SA-β-gal-positive cells and significantly reversed the ECH-induced reduction of endothelial senescence. Our results demonstrated that ECH employed endothelial injury and senescence in HUVECs via activation of the SIRT1 pathway.

Inhibition of ox-LDL-induced endothelial cell injury by LINC02381 knockdown through the microRNA-491-5p/transcription factor 7 axis.

Atherosclerosis (AS) is a complex multifactorial and chronic inflammatory vascular disease that contributes to the development of cardiovascular diseases. Abnormal cellular proliferation in human umbilical vein endothelial cells (HUVECs) is a crucial element in AS development. In this study, we investigated the potential role of the long noncoding RNA LINC02381/microRNA (miR)-491-5p/transcription factor 7 (TCF7) axis in regulating HUVEC injury in 30 participants suffering from AS and 30 healthy control participants. We established an in vitro model of AS in HUVECs using oxidized low-density lipoprotein (ox-LDL), and measured cellular mRNA and protein levels of LINC02381, miR-491-5p, and TCF7 in serum samples using reverse transcription-quantitative polymerase chain reaction and Western blotting assays. We evaluated cell viability, apoptosis, and inflammation using Cell Counting Kit-8, flow cytometry, and enzyme-linked immunosorbent assays, respectively. Moreover, we analyzed apoptosis-related protein expression using western blotting analysis and determined the association between miR-491-5p and LINC02381 or TCF7 using dual-luciferase reporter assay, RNA pull-down, and rescue experiments. We observed that LINC02381 was elevated, while miR-491-5p was downregulated in serum samples from participants with AS and in ox-LDL-treated HUVECs. LINC02381 knockdown was protective against HUVEC injury via miR-491-5p inhibition, which is its downstream target. Rescue experiments further demonstrated that miR-491-5p alleviated HUVEC injury by modulating TCF7. Thus, LINC02381 knockdown ameliorated HUVEC injury by regulating the miR-491-5p/TCF7 axis, which provides new insights into AS treatment strategies.

Alpha-solanine inhibits endothelial inflammation via nuclear factor kappa B signaling pathway.

Alpha-solanine (α-solanine) is the main glycoalkaloid in potato plants. It possesses anticarcinogenic properties and exerts toxic effects. Alpha-solanine can regulate the nuclear factor kappa B (NF-κB) signaling pathway in cancer cells and macrophages. However, little is known about the anti-inflammatory effects and the related molecular mechanisms of α-solanine on endothelial cells. This study aims to examine the effects of α-solanine on endothelial inflammation in vitro, and to evaluate its influence on regulating the NF-κB signaling pathway. Tumor necrosis factor alpha (TNF-α)-pcDNA3.1(+) plasmid vector was constructed and transfected into human umbilical vein endothelial cells (HUVECs). The expression of TNF-α was examined with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. Following treatment with α-solanine or the specific NF-κB inhibitor SN50 for 24 h, cell viability was detected using Cell Counting Kit-8 (CCK-8) assay. Interleukin 6 (IL-6) and TNF-α levels in cell supernatant were detected using enzyme-linked immunosorbent assay (ELISA). The relative protein levels of phospho-P65 (p-P65), phospho-inhibitor of NF-κBα (p-IκBα) and IκB kinase (IKK) α/β were examined with western blot. The α-solanine inhibits TNF-α-induced inflammatory injury in HUVECs. Compared with control cells, the cell viability was significantly decreased, the levels of TNF-α and IL-6 were significantly increased, and the relative protein levels of p-P65, p-IκBα and IKKα/β were significantly upregulated in TNF-α-overexpressed cells. The treatment with α-solanine or SN50 decreased the levels of TNF-α and IL-6, and downregulated the relative protein levels of p-P65, p-IκBα and IKKα/β in TNF-α-overexpressed HUVECs. This study demonstrated for the first time that α-solanine inhibits endothelial inflammation through the NF-κB signaling pathway. The α-solanine was suggested to be an inhibitor of the NF-κB signaling pathway in endothelial cells. The anti-inflammatory effect of α-solanine may provide a new perspective for the prevention and treatment of phlebitis.

Circular RNA circ_0026218 Suppressed Atherosclerosis Progression via miR-338-3p/SIRT6 Axis.

Multiple circular RNAs (circRNAs) are implicated in atherosclerosis (AS) pathogenesis. In fact, how circRNA 0026218 (circ_0026218) functions in AS remains unknown, and thus the functions and mechanisms of circ_0026218 in the injury of vascular endothelial cells are to be investigated. Microarray analysis was employed to screen out differentially expressed circRNAs in AS. A cell model was mimicked by treating Human umbilical vein endothelial cells (HUVECs) with oxidized low-density lipoprotein (ox-LDL). circ_0026218, microRNA-338-3p (miR-338-3p) and silent information regulator 6 (SIRT6) expressions in HUVECs with ox-LDL treatment were probed by qRT-PCR. The cell proliferative capabilities were exposed by CCK-8 assay. The contents of interleukin 6 (IL-6), interleukin 1β (IL-1β), and tumor necrosis factor α (TNF-α) were measured by ELISA. Oxidative stress kits were utilized to detect the levels of reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA). Flow cytometry was adopted to analyze the level of apoptosis of HUVECs. Dual-luciferase reporter gene assay and RIP assay were leveraged to expose the interplay between miR-338-3p and circ_0026218 or SIRT6 3'-UTR, respectively. In addition, the impacts of circ_0026216 and miR-338-3p on SIRT6 protein expressions were subjected to Western blot. circ_0026218 was greatly depleted in ox-LDL-stimulated HUVECs. circ_0026218 overexpression promoted viability of HUVECs in vitro and inhibited inflammatory response, oxidative stress, and apoptosis. circ_0026218 could adsorb miR-338-3p and positively modulated SIRT6 expressions via sponging miR-338-3p. Upregulation of this miRNA reversed the influence of circ_0026218 overexpression on ox-LDL-caused injury and apoptosis of HUVECs. Collectively, circ_0026218 upregulates SIRT6 expression through decoying miR-338-3p, thereby inhibiting ox-LDL-initiated injury of HUVECs. circ_0026218 is involved in the pathogenesis of AS.