Vascular Endothelial Cell Dysfunction Research Articles

Circ_0004104 knockdown alleviates oxidized low-density lipoprotein-induced dysfunction in vascular endothelial cells through targeting miR-328-3p/TRIM14 axis in atherosclerosis

BackgroundCircular RNAs have shown important regulatory roles in cardiovascular diseases, containing atherosclerosis (AS). We intended to explore the role of circ_0004104 in AS using oxidized low-density lipoprotein (ox-LDL)-induced vascular endothelial cells and its associated mechanism.MethodsReal-time quantitative polymerase chain reaction and Western blot assay were conducted to analyze RNA levels and protein levels, respectively. Cell viability, apoptosis, angiogenic ability and inflammatory response were assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, flow cytometry, capillary-like network formation assay and enzyme-linked immunosorbent assay, respectively. Cell oxidative stress was assessed using commercial kits. Dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA-pull down assay were performed to verify the intermolecular interaction.Resultsox-LDL exposure up-regulated the level of circ_0004104 in HUVECs. ox-LDL exposure suppressed cell viability and angiogenic ability whereas promoted the apoptosis, inflammation and oxidative stress of HUVECs partly through up-regulating circ_0004104. MicroRNA-328-3p (miR-328-3p) was confirmed as a target of circ_0004104. MiR-328-3p interference largely reversed circ_0004104 silencing-mediated effects in HUVECs upon ox-LDL exposure. MiR-328-3p interacted with the 3′ untranslated region of tripartite motif 14, and circ_0004104 positively regulated TRIM14 expression by sponging miR-328-3p. TRIM14 overexpression largely overturned miR-328-3p accumulation-induced influences in HUVECs upon ox-LDL exposure.ConclusionCirc_0004104 knockdown attenuated ox-LDL-induced dysfunction in HUVECs via miR-328-3p-mediated regulation of TRIM14.

Relationship Between Autophagy and Metabolic Syndrome Characteristics in the Pathogenesis of Atherosclerosis.

Atherosclerosis is the main cause of mortality in metabolic-related diseases, including cardiovascular disease and type 2 diabetes (T2DM). Atherosclerosis is characterized by lipid accumulation and increased inflammatory cytokines in the vascular wall, endothelial cell and vascular smooth muscle cell dysfunction and foam cell formation initiated by monocytes/macrophages. The characteristics of metabolic syndrome (MetS), including obesity, glucose intolerance, dyslipidemia and hypertension, may activate multiple mechanisms, such as insulin resistance, oxidative stress and inflammatory pathways, thereby contributing to increased risks of developing atherosclerosis and T2DM. Autophagy is a lysosomal degradation process that plays an important role in maintaining cellular metabolic homeostasis. Increasing evidence indicates that impaired autophagy induced by MetS is related to oxidative stress, inflammation, and foam cell formation, further promoting atherosclerosis. Basal and mild adaptive autophagy protect against the progression of atherosclerotic plaques, while excessive autophagy activation leads to cell death, plaque instability or even plaque rupture. Therefore, autophagic homeostasis is essential for the development and outcome of atherosclerosis. Here, we discuss the potential role of autophagy and metabolic syndrome in the pathophysiologic mechanisms of atherosclerosis and potential therapeutic drugs that target these molecular mechanisms.

Exosomal miR-512-3p derived from mesenchymal stem cells inhibits oxidized low-density lipoprotein-induced vascular endothelial cells dysfunction via regulating Keap1.

Atherosclerosis (AS) is a prevalent chronic inflammatory vascular disease. Upregulated oxidized low-density lipoprotein (ox-LDL) in the serum has been found to induce endothelial cells (ECs) apoptosis by increasing oxidative stress and promoting inflammatory response, which are essential mechanisms of AS development. Mesenchymal stem cells (MSCs), which secrete exosomes to transport microRNAs (miRNAs) and regulate cell functions, have become a research focus in recent years. The results of this study manifested that MSCs-derived exosomes were phagocytosed by EC. In addition, miR-512-3p enriched by MSCs- derived exosomes markedly inhibited ox-LDL-mediated EC damage, namely, accelerated EC proliferation, inhibited Caspase-3 activation and cell apoptosis, inhibited the levels of inflammatory cytokines (tumor necrosis factor-α, interleukin (IL)-1β, and IL-6) and oxidative factor MDA, and increased the contents of SOD and GSH-PX. Mechanistically, Keleh-like ECH-associated protein 1 (Keap1) was proved to be a functional target of miR-512-3p. Furthermore, silencing Keap1 limited ox-LDL-mediated EC cell dysfunction, while over-expressing Keap1 mitigated the exosomal miR-512-3p-mediated protective effect in Ox-LDL-induced EC. The above results confirmed that miR-512-3p shuttled by MSCs-derived exosomes protected EC against ox-LDL by targeting Keap1.

Anagliptin Protected against Hypoxia/Reperfusion-Induced Brain Vascular Endothelial Permeability by Increasing ZO-1.

Background and purpose: Cerebral ischemia-reperfusion injury is commonly induced during the treatment of ischemic stroke and is reported to be related to the blood–brain barrier destruction and brain vascular endothelial cell dysfunction. Anagliptin is a novel antidiabetic agent recently reported to protect neurons from oxidative stress. In the present study, we aim to investigate the protective property of anagliptin against oxygen–glucose deprivation and reperfusion (OGD/R)-induced injury on endothelial cells and clarify the potential underlying mechanism. Methods: OGD/R modeling was established on bEnd.3 brain endothelial cells. Cell viability was detected using the MTT assay, and the mitochondrial reactive oxygen species (ROS) level was measured using the mitoses red staining assay. The endothelial monolayer permeability was determined using an FITC-dextran permeation assay. The expression levels of NOX-4 and ZO-1 were evaluated using qRT-PCR and Western blot assays. The expressions of MLC-2, p-MLC-2, and myosin light chain kinase (MLCK) were determined using Western blot. Results: First, the decreased cell viability, upregulated NOX-4, and elevated mitochondrial ROS level in the endothelial cells induced by OGD/R were reversed by treatment with anagliptin. Second, the enlarged endothelial permeability and the decreased expression level of ZO-1 in the endothelial cells induced by OGD/R were alleviated by anagliptin. Third, the downregulation of ZO-1 and enlarged brain endothelial monolayer permeability induced by OGD/R were ameliorated by an MLCK inhibitor, ML-7. Lastly, the elevated expressions of MLCK and p-MLC-2 induced by OGD/R were suppressed by anagliptin. Conclusion: Anagliptin protected against hypoxia/reperfusion-induced brain vascular endothelial permeability by increasing the expression ZO-1, mediated by inhibition of the MLCK/MLC-2 signaling pathway.

Metabolic Reprogramming of Vascular Endothelial Cells: Basic Research and Clinical Applications.

Vascular endothelial cells (VECs) build a barrier separating the blood from the vascular wall. The vascular endothelium is the largest endocrine organ, and is well-known for its crucial role in the regulation of vascular function. The initial response to endothelial cell injury can lead to the activation of VECs. However, excessive activation leads to metabolic pathway disruption, VEC dysfunction, and angiogenesis. The pathways related to VEC metabolic reprogramming recently have been considered as key modulators of VEC function in processes such as angiogenesis, inflammation, and barrier maintenance. In this review, we focus on the changes of VEC metabolism under physiological and pathophysiological conditions.

TPTEP1 suppresses high glucose-induced dysfunction in retinal vascular endothelial cells by interacting with STAT3 and targeting VEGFA.

Diabetic retinopathy (DR) is a vascular complication of diabetes mellitus that causes visual impairment and blindness. Long noncoding RNAs (lncRNAs) have been revealed to be involved in biological processes of several diseases including DR. We designed this study to investigate the specific role of TPTEP1 in DR. First, we mimicked diabetic conditions with high glucose (HG) stimulation of human retinal vascular endothelial cells (HRVECs) and measured TPTEP1 expression in HG-stimulated HRVECs using RT-qPCR analysis. Then, CCK-8, Transwell, and Matrigel tube formation assays as well as western blot analysis were performed to reveal the biological functions of TPTEP1 in HG-stimulated HRVECs. Subsequently, bioinformatics analysis, RNA pull down, luciferase reporter and ChIP assays as well as western blot analysis evaluated the relationship of TPTEP1, signal transducer and activator of transcription 3 (STAT3) and vascular endothelial growth factor A (VEGFA) in HG-stimulated HRVECs. Finally, to verify the regulation of the TPTEP1/STAT3/VEGFA axis in HG-stimulated HRVECs, rescue experiments were carried out in HG-stimulated HRVECs. TPTEP1 presented a significant downregulation in HG-stimulated HRVECs. Additionally, TPTEP1 overexpression reduced viability, migration, and angiogenesis in HG-stimulated HRVECs. Moreover, TPTEP1 suppressed phosphorylation and nuclear translocation of STAT3, and thereby downregulated VEGFA mRNA and protein levels. Furthermore, TPTEP1 overexpression-mediated suppression of HG-induced dysfunction in HRVECs was countervailed by STAT3 upregulation or VEGFA upregulation. TPTEP1 alleviated HG-induced dysfunction in HRVECs via interacting with STAT3 and targeting VEGFA.

An Atherogenic Diet Disturbs Aquaporin 5 Expression in Liver and Adipocyte Tissues of Apolipoprotein E-Deficient Mice: New Insights into an Old Model of Experimental Atherosclerosis.

The dysfunction of vascular endothelial cells is profoundly implicated in the pathogenesis of atherosclerosis and cardiovascular disease, the global leading cause of death. Aquaporins (AQPs) are membrane channels that facilitate water and glycerol transport across cellular membranes recently implicated in the homeostasis of the cardiovascular system. Apolipoprotein-E deficient (apoE−/−) mice are a common model to study the progression of atherosclerosis. Nevertheless, the pattern of expression of AQPs in this atheroprone model is poorly characterized. In this study, apoE−/− mice were fed an atherogenic high-fat (HF) or a control diet. Plasma was collected at multiple time points to assess metabolic disturbances. At the endpoint, the aortic atherosclerotic burden was quantified using high field magnetic resonance imaging. Moreover, the transcriptional levels of several AQP isoforms were evaluated in the liver, white adipocyte tissue (WAT), and brown adipocyte tissue (BAT). The results revealed that HF-fed mice, when compared to controls, presented an exacerbated systemic inflammation and atherosclerotic phenotype, with no major differences in systemic methylation status, circulating amino acids, or plasma total glutathione. Moreover, an overexpression of the isoform AQP5 was detected in all studied tissues from HF-fed mice when compared to controls. These results suggest a novel role for AQP5 on diet-induced atherosclerosis that warrants further investigation.

The toxicity of ambient fine particulate matter (PM2.5) to vascular endothelial cells

AbstractSeveral epidemiologic and toxicological studies have widely regarded ambient fine particulate matter (PM2.5), the particles with an aerodynamic diameter less than 2.5 μm, as a strong potential threat to human health. PM2.5 exposure is mainly through the respiratory tract where it can permeate the lung alveoli and enter the blood circulation. After going into the circulation, PM2.5 directly confronts the vascular endothelial cells (VECs). The VECs, which are lined up in the innermost layer of the blood vessels, are essential in the homeostasis of physiological processes. Thus, the damage and dysfunction of VECs is a common cause of various diseases. In this review, we summarize the toxicity of PM2.5 to VECs including the pathophysiological mechanism and the effects of VEC‐mediated physiological functions. The review has discussed the association of impaired VEC function by PM2.5 with various diseases, indicating that the VECs may be an effective assessment of public health recommendations under PM2.5 exposure. Therefore, reducing the adverse effect of PM2.5 on VECs will prevent the potential occurrence and fatality of the relevant diseases in the future.

Retinal Vascular Endothelial Cell Dysfunction and Neuroretinal Degeneration in Diabetic Patients.

Diabetes mellitus (DM) has become a vital societal problem as epidemiological studies demonstrate the increasing incidence of type 1 and type 2 diabetes. Lesions observed in the retina in the course of diabetes, referred to as diabetic retinopathy (DR), are caused by vascular abnormalities and are ischemic in nature. Vascular lesions in diabetes pertain to small vessels (microangiopathy) and involve precapillary arterioles, capillaries and small veins. Pericyte loss, thickening of the basement membrane, and damage and proliferation of endothelial cells are observed. Endothelial cells (monolayer squamous epithelium) form the smooth internal vascular lining indispensable for normal blood flow. Breaking its continuity initiates blood coagulation at that site. The endothelium controls the process of exchange of chemical substances (nutritional, regulatory, waste products) between blood and the retina, and blood cell passing through the vascular wall. Endothelial cells produce biologically active substances involved in blood coagulation, regulating vascular wall tension and stimulating neoangiogenesis. On the other hand, recent studies have demonstrated that diabetic retinopathy may be not only a microvascular disease, but is a result of neuroretinal degeneration. Neuroretinal degeneration appears structurally, as neural apoptosis of amacrine and Muller cells, reactive gliosis, ganglion cell layer/inner plexiform (GCL) thickness, retinal thickness, and retinal nerve fiber layer thickness, and a reduction of the neuroretinal rim in minimum rim width (MRW) and functionally as an abnormal electroretinogram (ERG), dark adaptation, contrast sensitivity, color vision, and microperimetric test. The findings in early stages of diabetic retinopathy may precede microvascular changes of this disease. Furthermore, the article’s objective is to characterize the factors and mechanisms conducive to microvascular changes and neuroretinal apoptosis in diabetic retinopathy. Only when all the measures preventing vascular dysfunction are determined will the risk of complications in the course of diabetes be minimized.

Sulforaphane protects human umbilical vein endothelial cells from oxidative stress via the miR-34a/SIRT1 axis by upregulating nuclear factor erythroid-2-related factor 2.

Oxidative stress-induced vascular endothelial cell dysfunction serves an essential role in the initiation and development of atherosclerosis. Sulforaphane (SFN), a naturally occurring antioxidant, has previously demonstrated to exert protective effects on the endothelium against oxidative stress. However, further studies are required to determine its underlying molecular mechanism prior to clinical application. Accumulating evidence suggests that alterations in the microRNA (miRNA/miR)-34a/sirtuin-1 (SIRT1) axis occur with oxidative stress. Therefore, the present study aimed to investigate if SFN exerts a protective role against oxidative stress in vascular endothelial cells through regulation of the miR-34a/SIRT1 axis. Human umbilical vein endothelial cells (HUVECs) were treated with H2O2 in the presence or absence of SFN pretreatment. Cell viability and apoptosis were analyzed using CellTiter-Blue and flow cytometry, respectively. Reverse transcription-quantitative PCR and western blot analyses were performed to determine changes in the expression levels of miR-34a and SIRT1. The expression levels of miR-34a and SIRT1 were artificially regulated following transfection with miR-34a mimic and inhibitor or SIRT1expression plasmid and small interfering RNA, respectively. Subsequently, the effect of the expression changes of miR-34 and SIRT1 on oxidative stress-induced cell injury was investigated. Dual-luciferase reporter assay was used to confirm the targeted binding of miR-34a to SIRT1. SFN was found to ameliorate cellular damage caused by H2O2 and inhibited intracellular reactive oxygen species production. In addition, miR-34a upregulation was accompanied with reduced SIRT1 expression in HUVECs, following H2O2 treatment. miR-34a was revealed to directly target SIRT1 by binding to its 3'-untranslated region. Down-regulation of miR-34a and up-regulation of SIRT1 increased the survival of HUVECs under oxidative stress. Taken together, the results of the present study suggest that SFN may protect HUVECs from oxidative stress by inducing changes in the miR-34a/SIRT1 axis via upregulation of nuclear factor erythroid-2-related factor 2 expression.

Evidence of vascular endothelial dysfunction in Wooden Breast disorder in chickens: Insights through gene expression analysis, ultra-structural evaluation and supervised machine learning methods.

Several gene expression studies have been previously conducted to characterize molecular basis of Wooden Breast myopathy in commercial broiler chickens. These studies have generally used a limited sample size and relied on a binary disease outcome (unaffected or affected by Wooden Breast), which are appropriate for an initial investigation. However, to identify biomarkers of disease severity and development, it is necessary to use a large number of samples with a varying degree of disease severity. Therefore, in this study, we assayed a relatively large number of samples (n = 96) harvested from the pectoralis major muscle of unaffected (U), partially affected (P) and markedly affected (A) chickens. Gene expression analysis was conducted using the nCounter MAX Analysis System and data were analyzed using four different supervised machine-learning methods, including support vector machines (SVM), random forests (RF), elastic net logistic regression (ENET) and Lasso logistic regression (LASSO). The SVM method achieved the highest prediction accuracy for both three-class (U, P and A) and two-class (U and P+A) classifications with 94% prediction accuracy for two-class classification and 85% for three-class classification. The results also identified biomarkers of Wooden Breast severity and development. Additionally, gene expression analysis and ultrastructural evaluations provided evidence of vascular endothelial cell dysfunction in the early pathogenesis of Wooden Breast.

MiR-152-3p aggravates vascular endothelial cell dysfunction by targeting DEAD-box helicase 6 (DDX6) under hypoxia

ABSTRACT Stroke is a main cause of disability and death worldwide, and ischemic stroke accounts for most stroke cases. Recently, microRNAs (miRNAs) have been verified to play critical roles in the development of stroke. Herein, we explored effects of miR-152-3p on vascular endothelial cell functions under hypoxia. Human umbilical vein endothelial cells (HUVECs) were treated with hypoxia to mimic cell injury in vitro. Reverse transcription quantitative polymerase chain reaction revealed that miR-152-3p exhibited high expression in HUVECs treated with hypoxia. The inhibition of miR-152-3p reversed hypoxia-induced decrease in cell viability and the increase in angiogenesis, according to the results of cell counting kit-8 assays and tube formation assays. miR-152-3p inhibition reversed the increase in endothelial cell permeability mediated by hypoxia, as shown by endothelial cell permeability in vitro assays. In addition, the increase in protein levels of angiogenetic markers and the decrease in levels of tight junction proteins induced by hypoxia were reversed by miR-152-3p inhibition. Mechanistically, miR-152-3p directly targets 3ʹ-untranslated region of DEAD-box helicase 6 (DDX6), which was confirmed by luciferase reporter assays. DDX6 is lowly expressed in HUVECs under hypoxic condition, and mRNA expression and protein level of DDX6 were upregulated in HUVECs due to miR-152-3p inhibition. Rescue assays showed that DDX6 knockdown reversed effects of miR-152-3p on cell viability, angiogenesis and endothelial permeability. The results demonstrated that miR-152-3p aggravates vascular endothelial cell dysfunction by targeting DDX6 under hypoxia.

BRD4 and PIN1 gene polymorphisms are associated with high pulse pressure risk in a southeastern Chinese population

BackgroundBRD4 and PIN1 have been described to be involved in inflammation and vascular endothelial cell dysfunction, which in turn may increase pulse pressure.HypothesisGenetic mutations within the BRD4 and PIN1 genes could affect the risk of high pulse pressure.MethodsA total of four single nucleotide polymorphisms (SNPs) (BRD4: rs4808278; PIN1: rs2233678, rs2287838, and rs2233682) were genotyped in a cohort of 666 hypertensive patients and 232 normotensive controls with Chinese Han origin. Generalized multifactor dimensionality reduction (GMDR) was used to screen the best interaction combination among the four SNPs within the BRD4 and PIN1 genes and diabetes. Logistic regression analysis was performed to calculate the odds ratio (ORs) (95% confidence interval (CI)) for the association between the four SNPs.ResultsAdjusted for age, weight, waist circumference, drinking, smoking, hypertension, and diabetes, high pulse pressure risk was significantly higher for carriers with the rs4808278-TT genotype in BRD4 than those with wild genotypes (OR: 0.400, 95% CI: 0.217–0.737, P* < 0.05). However, we did not find any significant association of rs2233678, rs2287838, and rs2233682 in PIN1 with high pulse pressure susceptibility after covariate adjustment. GMDR analysis indicated a significant three-locus model (P = 0.0107) involving rs4808278, rs2233678, and diabetes, the cross-validation consistency of the three-locus models was 9/10, and the testing accuracy was 57.47%.ConclusionsGenetic mutations within BRD4 (rs4808278) could affect the susceptibility to high pulse pressure in a southeastern Chinese population.

Cilostazol improves hyperglycemia-induced impaired vasculo-angiogenesis through stimulating adiponectin/adiponectin receptor 1/Sirtuin 1 signaling pathway

Abstract Background Cilostazol is an antiplatelet agent with vasodilating effect working through increasing intracellular concentration of cyclic adenosine monophosphate (AMP). We and others have previously found that cilostazol has a favorable effect on vasculo-angiogenesis. However, there is no study to evaluate the effect of cilostazol on adiponectin and its receptors. Purpose This study investigated the effects of cilostazol on adiponectin/adiponectin receptors and Sirtuin 1 (Sirt1)/AMP-activated protein kinase (AMPK) signaling pathway for preventing high glucose (HG)-induced impaired vasculo-angiogenesis in vitro and in vivo. Methods and results Human umbilical vein endothelial cells (HUVECs), seeded onto Transwell insert, and human aortic smooth muscle cells (HASMCs), seeded onto 6-well plate at lower level, were co-cultured in HG condition (25 mM). Adiponectin concentrations in the supernatant of 6-welll-plate and Transwell insert were significantly higher when HASMCs were treated with cilostazol in a dose-response manner but not significantly changed when only HUVECs were treated with cilostazol. HG downregulated protein expression of adiponectin receptor-1 (adipoR1), adipoR2, and Sirt1 and phosphorylation of AMPKα1, whereas cilostazol treatment restored expression of adipoR1 and Sirt1 proteins and upregulated phosphorylation of AMPKα1 in HUVECs treated with HG but not adipoR2. The stimulating effect of cilostazol on AMPKα1 or Sirt1 was attenuated with Sirt1 or AMPKα1 gene knockdown, respectively. By using gene knockdown of adiponectin receptors or AMPKα1, or treatment of the Sirt1 inhibitor, our data showed that cilostazol prevented apoptosis, and stimulated proliferation, chemotactic motility and capillary-like tube formation in HG-treated HUVECs through adipoR1, AMPK, and Sirt1 signaling pathway but not adipoR2. Fifteen-week-old male ICR hyperglycemic mice, induced by streptozosin injection and high cholesterol diet feeding, were treated intraperitoneally with cilostazol (10 mg/kg) 2 times per day since day 1 to day 7 after hindlimb ischemia. Recovery of blood flow ratio (ipsilateral/contralateral) in the ischemic hindlimb 14–21 days after surgery and circulating CD34+CD45dim cells were significantly attenuated by adipoR1 knockdown but not adipoR2. Capillary density in the ischemic muscles was significantly lower in both adipoR1- and adipoR2-knockdown mice. Expression of Sirt1 as well as phosphorylation of AMPKα1/acetyl-CoA carboxylase and Akt/endothelial nitric oxide synthase in ischemic muscles were significantly attenuated by gene knockdown of adipoR1 or adipoR2. Conclusions Our data suggest that cilostazol prevents high glucose-induced endothelial dysfunction in vascular endothelial cells as well as enhances vasculo-angiogenesis in hyperglycemic mice by upregulation of adiponectin/adipoR2 and its downstream signaling molecules, Sirt1/AMPK. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): The Ministry of Health and Welfare, Executive Yuan, Taiwan; The Ministry of Science and Technology, Executive Yuan, Taiwan

Effects of DA-9801 on the inflammation and apoptosis induced by angiotensin II in human dermal microvascular endothelial cells

DA-9801, a plant-based drug used for the treatment of diabetic neuropathy, is known to improve angiotensin II (Ang II)-induced vascular endothelial cell dysfunction. However, the underlying mechanism is not fully understood. We aimed to determine whether the protective effect of DA-9801 against Ang II-induced endothelial cell dysfunction was mediated via inhibition of endothelial cell inflammation and apoptosis. Ang II-induced oxidative stress was attenuated by pretreatment of human dermal microvascular endothelial cells (HDMECs) with DA-9801. This prevented the Ang II-induced upregulation of NAD(P)H oxidase (the NOX4 and p22phox subunits) and reactive oxygen species. Further, pretreatment of HDMECs with DA-9801 ameliorated Ang II-mediated nuclear factor kappa B activity via prevention of the upregulation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase. It also decreased the Ang II-stimulated increase in inducible nitric oxide synthase (NOS) and decreased endothelial NOS protein expression. DA-9801 decreased Ang II-induced upregulation of intercellular adhesion molecule 1, vascular adhesion molecule, and E-selectin in HDMECs. Moreover, TUNEL and annexin V-FITC fluorescence staining for apoptosis and the activities of caspases 9, 7, and 3 decreased in HDMECs pretreated with DA-9801, indicating that the drug enhanced anti-apoptotic pathways. Thus, DA-9801 modulated Ang II-induced endothelial cell dysfunction via inflammatory and apoptotic pathways.

P38 and Casein Kinase 2 Mediate Ribonuclease 1 Repression in Inflamed Human Endothelial Cells via Promoter Remodeling Through Nucleosome Remodeling and Deacetylase Complex.

Vascular pathologies, such as thrombosis or atherosclerosis, are leading causes of death worldwide and are strongly associated with the dysfunction of vascular endothelial cells. In this context, the extracellular endonuclease Ribonuclease 1 (RNase1) acts as an essential protective factor in regulation and maintenance of vascular homeostasis. However, long-term inflammation causes strong repression of RNase1 expression, thereby promoting endothelial cell dysfunction. This inflammation-mediated downregulation of RNase1 in human endothelial cells is facilitated via histone deacetylase (HDAC) 2, although the underlying molecular mechanisms are still unknown. Here, we report that inhibition of c-Jun N-terminal kinase by small chemical compounds in primary human endothelial cells decreased physiological RNase1 mRNA abundance, while p38 kinase inhibition restored repressed RNase1 expression upon proinflammatory stimulation with tumor necrosis factor alpha (TNF-α) and poly I:C. Moreover, blocking of the p38 kinase- and HDAC2-associated kinase casein kinase 2 (CK2) by inhibitor as well as small interfering RNA (siRNA)-knockdown restored RNase1 expression upon inflammation of human endothelial cells. Further downstream, siRNA-knockdown of chromodomain helicase DNA binding protein (CHD) 3 and 4 of the nucleosome remodeling and deacetylase (NuRD) complex restored RNase1 repression in TNF-α treated endothelial cells implicating its role in the HDAC2-containing repressor complex involved in RNase1 repression. Finally, chromatin immunoprecipitation in primary human endothelial cells confirmed recruitment of the CHD4-containing NuRD complex and subsequent promoter remodeling via histone deacetylation at the RNASE1 promoter in a p38-dependent manner upon human endothelial cell inflammation. Altogether, our results suggest that endothelial RNase1 repression in chronic vascular inflammation is regulated by a p38 kinase-, CK2-, and NuRD complex-dependent pathway resulting in complex recruitment to the RNASE1 promoter and subsequent promoter remodeling.

Proteomics analysis of the protective effect of canola (Brassica campestris L.) bee pollen flavonoids on the tert-butyl hydroperoxide-induced EA.hy926 cell injury model

• Canola bee pollen flavonoids can protect against vascular endothelial cell injury. • Canola bee pollen flavonoids have antioxidant and anti-inflammatory effects on EA.hy926 cells. • iTRAQ proteomics technology was used for protection mechanism analysis. • The mechanism involves regulation of oxidative stress, lipid metabolism and mitochondrial function. Pollen contains various antioxidants, including flavonoids, which are beneficial to cardiovascular health. However, the protective effect and mechanism of pollen flavonoids extract on the cardiovascular system are still unclear. Herein, the protective effects of crude flavonoids of pollen (CFP) from canola ( Brassica campestris L. ) on tert-butyl hydroperoxide (TBHP)-induced cell injury were assessed in the EA.hy926 cell line. Then, the potential mechanism by which canola CFP protects vascular endothelial cells was explored at the proteome level and was verified by determining relevant indicators. Canola CFP exerted antioxidant and anti-inflammatory effects on the EA.hy926 cell injury model. Proteomics analysis and mechanism verification assays demonstrated that pretreatment with canola CFP produced effects primarily through maintenance of normal redox, mitochondrial function and lipid metabolism in cells. Our results indicate that canola CFP has great potential for preventing TBHP-induced vascular endothelial cell dysfunction.

Atorvastatin combined with low-dose dexamethasone for vascular endothelial cell dysfunction induced by chronic subdural hematoma.

Atorvastatin has been shown to be a safe and effective non-surgical treatment option for patients with chronic subdural hematoma. However, treatment with atorvastatin is not effective in some patients, who must undergo further surgical treatment. Dexamethasone has anti-inflammatory and immunomodulatory effects, and low dosages are safe and effective for the treatment of many diseases, such as ankylosing spondylitis and community-acquired pneumonia. However, the effects of atorvastatin and low-dose dexamethasone for the treatment of chronic subdural hematoma remain poorly understood. Hematoma samples of patients with chronic subdural hematoma admitted to the General Hospital of Tianjin Medical University of China were collected and diluted in endothelial cell medium at 1:1 as the hematoma group. Atorvastatin, dexamethasone, or their combination was added to the culture medium. The main results were as follows: hopping probe ion conductance microscopy and permeability detection revealed that the best dosages to improve endothelial cell permeability were 0.1 μM atorvastatin and 0.1 μM dexamethasone. Atorvastatin, dexamethasone, or their combination could markedly improve the recovery of injured endothelial cells. Mice subcutaneously injected with diluted hematoma solution and then treated with atorvastatin, dexamethasone, or their combination exhibited varying levels of rescue of endothelial cell function. Hopping probe ion conductance microscopy, western blot assay, and polymerase chain reaction to evaluate the status of human cerebral endothelial cell status and expression level of tight junction protein indicated that atorvastatin, dexamethasone, or their combination could reduce subcutaneous vascular leakage caused by hematoma fluid. Moreover, the curative effect of the combined treatment was significantly better than that of either single treatment. Expression of Krüppel-like factor 2 protein in human cerebral endothelial cells was significantly increased, as was expression of the tight junction protein and vascular permeability marker vascular endothelial cadherin in each treatment group compared with the hematoma stimulation group. Hematoma fluid in patients with chronic subdural hematoma may damage vascular endothelial cells. However, atorvastatin combined with low-dose dexamethasone could rescue endothelial cell dysfunction by increasing the expression of tight junction proteins after hematoma injury. The effect of combining atorvastatin with low-dose dexamethasone was better than that of atorvastatin alone. Increased expression of Krüppel-like factor 2 may play an important role in the treatment of chronic subdural hematoma. The animal protocols were approved by the Animal Care and Use Committee of Tianjin Medical University of China on July 31, 2016 (approval No. IRB2016-YX-036). The study regarding human hematoma samples was approved by the Ethics Committee of Tianjin Medical University of China on July 31, 2018 (approval No. IRB2018-088-01).

Microparticles-Mediated Vascular Inflammation and its Amelioration by Antioxidant Activity of Baicalin.

Microparticles (MPs) are extracellular vesicles (0.1–1.0 μm in size), released in response to cell activation or apoptosis. Endothelial microparticles (EC-MP), vascular smooth muscle cell microparticles (VSMC-MP), and macrophage microparticles (MØ-MP) are key hallmarks of atherosclerosis progression. In our current study, we investigated the potent antioxidant activity of baicalin to ameliorate MP-induced vascular smooth muscle cell (VSMC) dysfunction and endothelial cell (EC) dysfunction, as well as the production of inflammatory mediators in macrophage (RAW264.7). In our study, baicalin suppressed the apoptosis, reactive oxygen species (ROS) generation, NO production, foam cell formation, protein expression of inducible nitric oxide synthase and cyclooxygenase-2 in MØ-MP-induced RAW264.7. In addition, VSMC migration induced by VSMC-MP was dose-dependently inhibited by baicalin. Likewise, baicalin inhibits metalloproteinase-9 expression and suppresses VSMC-MP-induced VSMC proliferation by down-regulation of mitogen-activated protein kinase and proliferating cell nuclear antigen protein expressions. Baicalin also inhibited ROS production and apoptosis in VSMC. In EC, the marker of endothelial dysfunction (endothelial senescence, upregulation of ICAM, and ROS production) induced by EC-MP was halted by baicalin. Our results suggested that baicalin exerts potent biological activity to restore the function of EC and VSMC altered by their corresponding microparticles and inhibits the release of inflammation markers from activated macrophages.

TMEM98, a novel secretory protein, promotes endothelial cell adhesion as well as vascular smooth muscle cell proliferation and migration.

Transmembrane protein 98 (TMEM98) is a novel gene, and its function has not been well investigated. In a prior study, we have shown that siRNA-mediated knockdown of TMEM98 inhibited interleukin-8 (IL-8) promoted endothelial cell (EC) adhesion, as well as vascular smooth muscle cell (VSMC) proliferation and migration in the vascular endothelial and smooth muscle cell dysfunction. Herein, we used gain- and loss-of-function approaches combined with biochemical techniques to further explore the role of TMEM98 in the vascular wall cell. The expression and secretion of TMEM98 was increased in cultured human umbilical vein endothelial cells (HUVECs) and VSMCs treated with IL-8 and platelet-derived growth factor-BB (PDGF-BB). Also, PDGF-BB secretion was increased in TMEM98-treated HUVECs and VSMCs. Thus, it appears that TMEM98 and PDGF-BB form a positive feedback loop in potentiation of EC adhesion, as well as VSMC proliferation and migration. Knockdown of TMEM98 mediated by siRNA inhibited PDGF-BB-promoted EC adhesion by downregulating the expression of ICAM-1 and VCAM-1, as well as impaired the proliferation and migration of VSMCs by suppressing the AKT/GSK3β/cyclinD1 signaling pathway and reducing the expression of β-catenin. Hence, TMEM98 promoted EC adhesion by inducing the expression of ICAM-1/VCAM-1 and triggered VSMC proliferation and migration by activating the ERK and AKT/GSK3β signaling pathways. Taken together, TMEM98 may serve as a potential therapeutic target for the clinical treatment of vascular endothelial and smooth muscle cell dysfunction.