Endothelial Cell Inflammation Research Articles

The endoplasmic reticulum (ER) chaperone GRP78 is secreted during ER Stress and alleviates endothelial cell inflammation

Abstract Introduction Glucose-Regulated Protein 78kD (GRP78) is a chaperone and the main regulator of the ER-stress response which is triggered by a variety of conditions that disturb folding of proteins in the ER. Upon ER Stress, GRP78 activates the unfolded protein response (UPR), which aims to clear unfolded proteins and restore ER homeostasis. Recently, extracellular secretion of GRP78 was described. However, the pathophysiological relevance of secreted GRP78 in atherosclerosis and endothelial cell inflammation remains to be elucidated. Aim of this study is to investigate the role of GRP78 secretion in endothelial cells. Methods and Results First, we sought to investigate if vascular cells secrete GRP78 during ER Stress. Human coronary artery endothelial cells (HCAEC) were treated with the ER stress inductor tunicamycin for up to 48h. After ER Stress induction, Western Blot and ELISA experiments detected an increased intracellular GRP78 expression. Intriguingly, prolonged ER Stress also promoted extracellular secretion of GRP78. Unbiased proteomic analysis of the HCAEC secretome confirmed that after ER-Stress induction, GRP78 is one of the most highly upregulated extracellular proteins (2.43-fold). Co-incubation with Brefeldin A, an inhibitor of ER-Golgi protein transport, abolished extracellular secretion (Fig.1). Hence, ER-Stress-induced GRP78 secretion is an actively regulated process. Next, the effect of GRP78 containing conditioned medium (CM) on HCAEC was analyzed. For control, HCAEC were co-incubated with BFA, or GRP78-knockdown was performed by siRNA. Treatment with GRP78 containing CM decreased GRP78 mRNA expression in target cells (0.35-fold vs. control [+BFA], p<0.0001). Furthermore, it increased viability ( BFA: 93.0 % vs. 83%, p=0.002; GRP78-siRNA: 94% vs. 85%, p=0.01) and decreased the rate of apoptosis (e.g. BFA: 1.7-fold vs. 5.1, p=0.02). Moreover, expression of markers of vascular inflammation and ER Stress (e.g., NF-κB and CHOP) were decreased when compared to CM without GRP78 (Fig.2). Furthermore, increased H2O2-generation was measured (BFA: 1.4-fold, p>0.001, GRP78-siRNA:1.3-fold, p=0.004) (Fig. 2). To confirm that the beneficial effects of the CM are in fact caused by GRP78, we treated HCAEC with recombinant GRP78 and observed an increased cell viability in a dose-dependent manner (1000 ng/ml: p=0.02 vs. control). CRIPTO is a membrane-bound co-receptor for growth factors and was previously linked to GRP78-signalling. siRNA-Knockdown of CRIPTO reduces viability and leads to cell apoptosis, irrespective of GRP78-treatment. Thus, GRP78-mediated effects may be mediated by CRIPTO. Conclusion Endothelial ER Stress promotes GRP78 secretion. Presence of GRP78 in conditioned medium ameliorates subsequent ER Stress and endothelial inflammation, which play a critical role in atherogenesis. Further investigations are important to shed light on the mechanism of release and the effect on target cells.

Intravenous iron administration in patients with heart failure alters endothelial homeostasis with early release of endothelial microvesicles and subsequent mobilization of endothelial progenitor cell

Abstract Background Intravenous iron supplementation is an established therapy for patients with heart failure (HF) with reduced ejection fraction (LV-EF<50%) and iron deficiency, resulting in reduced risk of HF hospitalization. Since iron may induce oxidative stress, inflammation and apoptosis in endothelial cells, concerns persist regarding potential adverse vascular effects of iron therapy. To evaluate endothelial health following ferric carboxymaltose (FCM) administration, we assessed the profile of circulating endothelial microvesicles (EMVs) and endothelial progenitor cells (EPCs) in a cohort of 23 HF patients using flow cytometry. Results Compared to healthy control subjects (HC; n=15), baseline levels of CD31+/CD41- EMVs were significantly higher and EMVs featured a more apoptotic phenotype in HF patients. Following administration of 1000mg FCM, EMV levels showed an early but transient increase (Fig. 1) and displayed an altered phenotype profile with dominant augmentation of EMVs expressing the inducible markers CD62E and CD54, indicating endothelial activation and injury. Levels of circulating vasoregenerative CD45lowCD34+KDR+ EPCs were lower in HF patients and FCM application resulted in an early decrease of EPCs followed by substantial mobilization into the circulation after one week (Fig. 2). Levels of EMVs and EPCs returned to baseline values within two and four weeks, respectively. Compared to HF patients with preserved renal function, the EMV/EPC ratio was higher in HF patients whereas EPC mobilization was diminished with additional chronic kidney disease, suggesting impaired vascular repair capacity. In vitro experiments with cultured endothelial cells revealed that FCM at therapeutic concentrations dose-dependently promotes endothelial apoptosis, increases expression of adhesion molecules and CXCL12, and triggers the generation of EMVs. Conclusion Intravenous iron supplementation with FCM in HF patients provokes a biphasic response with early release of CD62E+ and CD54+ enriched EMVs and subsequent mobilization of EPCs, suggesting temporary aggravation of endothelial dysfunction upon FCM supplementation and consecutive engagement of a vascular defense program.

Abstract 16881: Endothelial KLF11 is a Novel Endogenous Protectant Against Diabetic Atherosclerosis

Background: Atherosclerosis is the leading cause of cardiovascular diseases, which is also the primary cause of mortality among diabetic patients. Endothelial cell (EC) dysfunction is a critical early step in the development of atherosclerosis and aggravated in the presence of concurrent diabetes. Krüppel like factor 11 (KLF11), a transcription factor involved in diabetes, inhibits EC inflammation via regulation of NF-κB signaling. However, the potential function of KLF11 in pathogenesis of diabetic atherosclerosis remains undefined. Methods and results: Diet-induced type 2 diabetic atherosclerosis model is established in LDLR -/- mice by feeding a diabetogenic high-fat diet with cholesterol (DDC). KLF11 expression was increased in ECs from ascending aorta of LDLR -/- mice fed DDC diet. Moreover, EC specific KLF11 gain- (transgenic) and loss-of-function (knockout) mice in a LDLR -/- background were fed a DDC for 16 weeks. Endothelial KLF11 deficiency (Klf11 ECKO -LDLR -/- ) markedly increased atherogenesis, while EC-selective KLF11 overexpression significantly inhibited DDC-induced atherogenesis. Single cell RNA sequencing profiling the ECs isolated from DDC-fed Klf11 ECKO -LDLR -/- mice and littermate controls showed that KLF11 depletion increased endothelial-to-mesenchymal transition (EndMT) during atherogenesis. In vitro studies showed that KLF11 not only inhibited EC inflammation and oxidative stress, but also diminished EndMT in response to high glucose. Conversely, knockdown of KLF11 augmented the EC dysfunction and EndMT. Conclusions: Our study indicates that endothelial KLF11 protects against diabetic atherosclerosis through inhibition of EC inflammation, oxidative stress and EndMT.

CREB1 Silencing Protects Against Inflammatory Response in Rats with Deep Vein Thrombosis Through Reducing RPL9 Expression and Blocking NF-κB Signaling.

Apoptosis and inflammation of vascular endothelial cells (VECs) are the most important causes of deep vein thrombosis (DVT). cAMP response element binding protein 1 (CREB1) encodes a transcription factor that binds as a homodimer to the cAMP-responsive element and can promote inflammation. CREB1 is found to be upregulated in the plasma of patients with venous thromboembolism. However, the biological functions of CREB1 in DVT remain unknown. We evaluated the effect of CREB1 in a rat model of inferior vena cava (IVA) stenosis-induced DVT. IVC stenosis resulted in stable thrombus, inflammatory response and CREB1 upregulation, whereas CREB1 knockdown inhibited thrombus and inflammation in DVT rats. In vitro analysis showed that CREB1 knockdown inhibited VEC apoptosis. Mechanistically, CREB1 knockdown reduced Ribosomal protein L9 (RPL9) expression and blocked the NF-κB pathway. Therefore, CREB1 may become a potential therapeutic target of DVT prevention.

Visualizing the spatial distribution of inflammation in the depressed brain with a targeted MRI nanoprobe in vivo

Depression is a prevalent mental illness that imposes a substantial public health burden. However, the diverse clinical phenotypes observed in patients make it difficult to realize precise diagnosis. Recently, accumulating preclinical and clinical evidence has suggested that inflammation is involved in the pathophysiology of depression. Herein, a molecular imaging–based strategy was proposed as a means to diagnose depression precisely by specifically visualizing the inflammation status associated with depression. Inflammation-targeting MRI nanoprobes were constructed by attaching an intercellular cell adhesion molecule-1 (ICAM-1)-targeting peptide to biocompatible Fe3O4 nanoparticles. Systematic studies demonstrated that the nanoprobes could specifically target inflamed vascular endothelial cells and visualize the spatial distribution of inflammation in the depressed brain in vivo through susceptibility-weighted imaging (SWI), which was further confirmed by histological analysis. Additionally, these inflammatory brain regions identified by nanoprobe-based imaging are consistent with the focal regions closely associated with the symptoms of depression as reported in previous behavioral studies. Overall, this is the first study to directly visualize the distribution of inflammation in the depressed brain in vivo through a molecular imaging strategy, which may not only facilitate insight into the biological mechanism underlying depression but also provide a potential target within the depressed brain for the further development of anti-inflammatory therapies.

HMGB1 Induced Oxidative Stress and Inflammation in Endothelial Cells Exposed to Impinging Flow

Introduction: Oxidative stress and inflammation contribute to many aspects of the pathological processes involved in intracranial aneurysm (IA). However, the underlying mechanism for inducing oxidative stress and inflammation under impinging flow remains unclear. Accumulating evidence has shown that high mobility group box-1 (HMGB1) is associated with oxidative stress-related chronic diseases and inflammatory responses. Therefore, we aimed to investigate whether HMGB1 is involved in oxidative stress and inflammatory responses in endothelial cells (ECs) exposed to impinging flow. Methods: We used a modified T-chamber to simulate the in vitro situation of human umbilical vein ECs (HUVECs) subjected to impinging flow at the arterial bifurcation in order to analyze the effect of wall shear stress (WSS) on the ECs. To investigate the role of HMGB1 in this process, we transfected ECs with short hairpin RNA (shRNA) before conducting impinging flow experiments. Intracellular reactive oxygen species (ROS) were measured by flow cytometry, and malondialdehyde, glutathione, and superoxide dismutase levels were measured to assess oxidative stress. Inflammation was assessed by measuring the mRNA expression levels of IL-1β, IL-6, and IL-8 using reverse transcription-quantitative polymerase chain reaction. We also examined the cellular localization of HMGB1 by immunofluorescence. Results: Exposure of HUVECs to WSS can increase the level of oxidative stress and inflammatory response. WSS increased the expression of HMGB1 in ECs and promoted the translocation of HMGB1 from cytosol to cytoplasm. When we knocked down HMGB1, the level of oxidative stress and inflammatory response caused by WSS in ECs decreased, suggesting that HMGB1 can mediate the oxidative stress and inflammatory response in HUVECs exposed to WSS. Conclusions: HMGB1 induced oxidative stress and inflammatory response in ECs exposed to impinging flow.

A novel circular RNA, circSQSTM1, protects the endothelial function in atherosclerosis

A novel circRNA named circSQSTM1 (hsa_circRNA_075320) was screened out in atorvastatin (ATV) stimulated endothelial cells (ECs) by our group. Considering the anti-atherosclerotic function of ATV, we hypothesized the circSQSTM1 could protect ECs functions in AS progression. The effects of circSQSTM1 on ECs inflammation, oxidative stress and autophagy were measured by qRT-PCR, Western blotting, monocyte-endothelial adhesion assay, dichloro-dihydro-fluorescein diacetate and mCherry-GFP-LC3 labeling. A luciferase reporter assay, RNA immunoprecipitation, MS2-tagging system and fluorescence in situ hybridization were performed to identify the biological functions of circSQSTM1. The partial left carotid artery ligation model and atherosclerosis model were established to analyze the effects of circSQSTM1 on atherosclerosis progression in vivo. Our results revealed that ATV induced the accumulation of circSQSTM1 in ECs via suppressing m6A modified degradation. In the cytoplasm, circSQSTM1 could relieve Sirt1 by competitively sponging miR-23b-3p. In the nucleus, circSQSTM1 directly interacts with eIF4A3 and promoting the efficient nuclear export of FOXO1 mRNA, which encodes FOXO1 transcription factor to directly activate Sirt1 promoter activity. Hence, circSQSTM1 reduced inflammation, inhibited oxidative stress and promoted autophagy by upregulating Sirt1 in ECs. Moreover, circSQSTM1 overexpression in ECs attenuated the progression of atherosclerosis in ApoE−/− mice. Taken together, the unique noncoding RNA known as circSQSTM1 took a protective role to the ECs in atherosclerosis.

Mitochondrial DNA is a key driver in cigarette smoke extract-induced IL-6 expression.

Smoking is an independent risk factor for atherosclerosis, the primary pathogenesis of which is inflammation. We recently reported that cigarette smoke extract (CSE) causes cytosolic and extracellular accumulation of both nuclear (n) and mitochondrial (mt) DNA, which leads to inflammation in human umbilical vein endothelial cells (HUVECs). In this study, we examined whether inflammation induction depends more on cytosolic nDNA or mtDNA, and which chemical constituents of CSE are involved. Acrolein (ACR), methyl vinyl ketone (MVK), and 2-cyclopenten-1-one (CPO) were used in the experiments, as these are the major cytotoxic factors in CSE in various cell types. Stimulation with ACR, MVK, or CPO alone resulted in the accumulation of DNA double-strand breaks (DSBs), but not oxidative DNA damage, accumulation of cytosolic DNA, or increased expression of inflammatory cytokines. Simultaneous administration of all three constituents (ALL) resulted in oxidative DNA damage in both the nucleus and mitochondria, accumulation of DSBs, reduced mitochondrial membrane potential, induction of minority mitochondrial outer membrane permeabilization, accumulation of cytosolic free DNA, and increased expression of inflammatory cytokines such as IL-6 and IL-1α. Treatment with N-acetyl-L-cysteine, a reactive oxygen species scavenger, suppressed oxidative DNA damage and the increased expression of IL-6 and IL-1α induced by ALL or CSE. The ALL- or CSE-induced increase in IL-6 expression, but not that of IL-1α, was suppressed by mtDNA depletion. In conclusion, ACR, MVK, and CPO may strongly contribute to CSE-induced inflammation. More importantly, cytosolic free mtDNA is thought to play an important role in IL-6 expression, a central mediator of inflammation.

Dendritic Cell-Derived Exosomes Stimulated by Treponema pallidum Induce Endothelial Cell Inflammatory Response through the TLR4/MyD88/NF-κB Signaling Pathway.

Exosomes have been implicated in vascular damage in recent research. The influence of dendritic cell-derived exosomes generated by Treponema pallidum (T. pallidum) on the inflammatory process of vascular cells was examined in this study. Human umbilical vein endothelial cells (HUVECs) were cocultured with exosomes isolated from dendritic cells induced by T. pallidum. Western blot and reverse transcription-quantitative real-time polymerase chain reaction were used to assess toll-like receptor 4 (TLR4) expression and the quantity of proinflammatory cytokines. The findings showed that the expression of TLR4 was considerably upregulated, and TLR4 knockdown dramatically reduced interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) production in exosome-treated HUVECs. Furthermore, TLR4 silencing reduced myeloid differentiation primary response protein 88 (MyD88) and nuclear factor kappa light chain enhancer of activated B cells (NF-κB) levels in exosome-treated HUVECs. Additionally, suppression of the activity of NF-κB with BAY11-7082, an NF-κB inhibitor, also reduced the exosome-treated inflammatory response. Our results suggested that dendritic cell-derived exosomes stimulated by T. pallidum induced endothelial cell inflammation, and the TLR4/MyD88/NF-κB signal axis was activated, significantly increasing IL-1β, IL-6, and TNF-α expression. This may have a significant role in the vascular inflammatory response in syphilis, which would contribute to the understanding of the pathogenesis of syphilis and the host immunological response to T. pallidum.

Glycyl–tRNA Synthetase Induces Psoriasis-Like Skin by Facilitating Skin Inflammation and Vascular Endothelial Cell Angiogenesis

Psoriasis is characterized by excessive keratinocyte proliferation and immunocyte infiltration, but the underlying pathogenesis remains unclear. Aminoacyl-tRNA synthetases are universally expressed enzymes that catalyze the first step of protein synthesis. Glycyl-tRNA synthetase (GARS) is a member of the aminoacyl-tRNA synthetase family. In addition to its canonical function, we found that GARS was overexpressed in the serum and skin lesions of patients with psoriasis. Moreover, GARS was highly expressed in human skin keratinocytes, and GARS knockdown in keratinocytes suppressed cell proliferation and promoted apoptosis through NF-κB/MAPK signaling pathway. Moreover, intradermal injection of recombinant GARS protein caused skin thickening, angiogenesis, and IFN/TNF-driven skin inflammation. Intriguingly, the reported functional receptor for GARS, cadherin 6 (CDH6), was specifically expressed in vascular endothelial cells, and we found that keratinocyte-derived GARS promotes inflammation and angiogenesis of vascular endothelial cells through CDH6. In addition, intradermal injection of GARS aggravated the phenotype and angiogenesis in imiquimod-induced psoriasiform dermatitis models, whereas the psoriatic phenotype and angiogenesis were relieved after knockdown of GARS by adeno-associated virus. Taken together, the results of this study identify the critical role of GARS in the pathogenesis of psoriasis and suggest that blocking GARS may be a therapeutic approach for alleviating psoriasis.

SIRT6 Protects Against Lipopolysaccharide-Induced Inflammation in Human Pulmonary Lung Microvascular Endothelial Cells.

Inflammatory response in the pulmonary endothelium drives the pathogenesis of acute lung injury and sepsis. Sirtuin 6 (SIRT6), a member of class III NAD+-dependent deacetylases belonging to the sirtuin family, regulates senescence, metabolism, and inflammation and extends lifespan in mice and model organisms. However, the role of SIRT6 in pulmonary endothelial inflammation is unknown. Thus, we hypothesized that SIRT6 suppresses inflammatory response in human lung microvascular cells (HLMEC) and ensues monocyte adhesion to endothelial cells. Primary HLMECs were treated with control or SIRT6 adenovirus or SIRT6 agonist, with or without lipopolysaccharide (LPS) treatment. We observed that treatment with LPS did not affect the protein expression of SIRT6 in HLMECs. However, adenovirus-mediated SIRT6 overexpression attenuated LPS-induced VCAM1 gene and protein expression, followed by decreased monocyte adhesion to endothelial cells. Similarly, activation of SIRT6 by a recently reported SIRT6 activator UBCS039, but not the regioisomer negative control compound UBCS060, ameliorated LPS-induced VCAM1 mRNA and protein expression as well as monocyte adhesion. Moreover, luciferase assay revealed that SIRT6 adenovirus decreased the activity of NF-κB, the master regulator of vascular inflammation. Taken together, these results indicate that molecular and pharmacological activation of SIRT6 protects against lung microvascular inflammation via suppressing NF-κB activation, implicating the therapeutic potential of the SIRT6 activators for lung disorders associated with microvascular inflammation.

Exosome-derived circ_0001785 delays atherogenesis through the ceRNA network mechanism of miR-513a-5p/TGFBR3

PurposeEndothelial cell dysfunction is a major cause of early atherosclerosis. Although the role of extracellular vesicles in stabilizing atherosclerotic plaques is well established, the effect of circulating exosomes on plaque formation is still unknown. Here, we explored the effect of exosomes on atherosclerosis based on the function that exosomes can act on intercellular communication.Patients and methodsWe extracted serum exosomes from the blood of CHD patients (CHD-Exo) and healthy individuals (Con-Exo). The obtained exosomes were co-cultured with human umbilical vein endothelial cells (HUVECs) in vitro. In addition, we determined that circ_0001785 functions as a competing endogenous RNA (ceRNAs) in coronary artery disease by dual luciferase reporter gene analysis. The protective effect of circ_0001785 against endothelial cell injury was also verified using over-expression lentiviral transfection functional assays. In vivo experiments, we injected over-expressed circ_0001785 lentivirus into the tail vein of mice to observe its therapeutic effect on a mouse model of atherosclerosis.ResultsThe vitro co-cultured results showed that the amount of plasma-derived exosomes have an increase in patients with coronary artery disease, and the inflammation and apoptosis of endothelial cells were exacerbated. Over-expression of circ_0001785 reduced endothelial cell injury through the ceRNA network pathway of miR-513a-5p/TGFBR3. Quantitative reverse transcription-polymerase chain reaction identified that the expressed amount of circ_0001785 was reduced in the circulating peripheral blood of CHD patients and increased within human and mouse atherosclerotic plaque tissue. The results of in vivo experiments showed that circ_0001785 reduced aortic endothelial cell injury and the formation of intraplaque neo-vascularization, and enhanced left ventricular diastolic function, thereby delaying the development of atherosclerosis in mice.ConclusionOur results demonstrated a new biomarker, exosome-derived circ_0001785, for atherogenesis, which can reduce endothelial cell injury and thus delay atherogenesis through the miR-513a-5p/TGFBR3 ceRNA network mechanism, providing an exosome-based intervention strategy for atherosclerosis.

Heat stress combined with lipopolysaccharide induces pulmonary microvascular endothelial cell glycocalyx inflammatory damage in vitro.

Heat stroke is a life-threatening disease with high mortality and complications. Endothelial glycocalyx (EGCX) is essential for maintaining endothelial cell structure and function as well as preventing theadhesion of inflammatory cells. Potential relationship that underlies the imbalance in inflammation and coagulation remains elusive. Moreover, the role of EGCX in heat stroke-induced organ injury remained unclear. Therefore, the current study aimed to illustrate if EGCX aggravates apoptosis, inflammation, and oxidative damage in human pulmonary microvascular endothelial cells (HPMEC). Heat stress and lipopolysaccharide (LPS) were employed to construct in vitro models to study the changes of glycocalyx structure and function, as well as levels of heparansulfate proteoglycan (HSPG), syndecan-1 (SDC-1), heparansulfate (HS), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, Von Willebrand factor (vWF), endothelin-1 (ET-1), occludin, E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and reactive oxygen species (ROS). Here, we showed that heat stress and LPS devastated EGCX structure, activated EGCX degradation, and triggered oxidative damage and apoptosis in HPMEC. Stimulation of heat stress and LPS decreased expression of HSPG, increased levels of SDC-1 and HS in culture supernatant, promoted the production and release of proinflammation cytokines (TNF-α and IL-6,) and coagulative factors (vWF and ET-1) in HPMEC. Furthermore, Expressions of E-selection, VCAM-1, and ROS were upregulated, while that of occludin was downregulated. These changes could be deteriorated by heparanase, whereas they meliorated by unfractionated heparin. This study indicated that EGCX may contribute to apoptosis and heat stroke-induced coagulopathy, and these effects may have been due to the decrease in the shedding of EGCX.

The Contribution of Serum Sialic Acid Binding Immunoglobulin-Like Lectin 1(sSIGLEC-1) as an IFN I Signature Biomarker in the Progression of Atherosclerosis in Egyptian Systemic Lupus Erythematosus (SLE) Patients.

Clinical symptoms of systemic lupus erythematosus (SLE), such as atherosclerosis and related cardiovascular diseases, are caused by inflammatory cytokines and endothelial cell damage. The serum sialic acid binding immunoglobulin-like lectin 1 (sSIGLEC-1) is thought to be an alternative biomarker of IFN signature and may have a role in the pathogenesis of atherosclerosis. The aim of the study was to measure the levels of sSIGLEC-1 in the serum of SLE patients in comparison to a control group and examine the associations between sSIGLEC-1, SLEDAI, lipid profile, oxidized low density lipoprotein (oxLDL), and carotid intima media thickness (CIMT) to investigate whether sSIGLEC-1 participates in the development of atherosclerosis. sSIGLEC-1 levels were tested in 53 patients and 20 volunteers using ELISA kit. Duplex measurements were performed on all subjects to measure CIMT. SLE patients had significantly higher values of sSIGLEC-1 (P < 0.0001), total cholesterol (P = 0.029), triglycerides (P = 0.001), low density lipoprotein (P = 0.032), oxLDL (P = 0.001), right CIMT (P = 0.0099) and a significantly lower value of high-density lipoprotein (P = 0.04) when compared to controls. sSIGLEC-1 had significant positive correlations with right CIMT (r = 0.5, P < 0.0002) and oxLDL (r = 0.67, P < 0.0001) in all SLE patients. When compared to non-dyslipidemic patients, the dyslipidemic group exhibited significantly higher levels of all previous parameters except HDL and left CIMT. Circulating form of SIGLEC-1 accelerates atherosclerosis and provides a simple way to predict the occurrence of atherosclerosis in SLE patients.

Inflammatory Cell-Derived MYDGF Attenuates Endothelial LDL Transcytosis to Protect Against Atherogenesis.

Inflammation contributes to the pathogenesis of atherosclerosis. But little is known about the potential benefits of inflammatory cells to atherosclerosis. The aim of this study was to investigate the function of inflammatory cells/endothelium axis and determine whether and how inflammatory cell-derived MYDGF (myeloid-derived growth factor) inhibited endothelial LDL (low-density lipoprotein) transcytosis. In in vivo experiments, both loss- and gain-of-function strategies were used to evaluate the effect of inflammatory cell-derived MYDGF on LDL transcytosis. We generated monocyte/macrophage-targeted MYDGF-null mice on an Ldlr (LDL receptor)-/- background in the loss-of-function strategy and restored the inflammatory cell-derived MYDGF by bone marrow transplantation and inflammatory cell-specific overexpression of MYDGF mice model in the gain-of-function strategy. In in vitro experiments, coculture experiments between primary mouse aortic endothelial cells and macrophages and mouse aortic endothelial cells supplemented with or without recombinant MYDGF were conducted. Inflammatory cell-derived MYDGF deficiency aggravated endothelial LDL transcytosis, drove LDL uptake by artery wall, and thus exacerbated atherosclerosis in vivo. Inflammatory cell-derived MYDGF restoration by bone marrow transplantation and inflammatory cell MYDGF overexpression alleviated LDL transport across the endothelium, prevented LDL accumulation in the subendothelial space, and subsequently ameliorated atherosclerosis in vivo. Furthermore, in the in vitro study, macrophages isolated from MYDGF+/+ mice and recombinant MYDGF attenuated LDL transcytosis and uptake in mouse aortic endothelial cells. Mechanistically, MYDGF inhibited MAP4K4 (mitogen-activated protein kinase kinase kinase kinase isoform 4) phosphorylation, enhanced activation of Akt (protein kinase B)-1, and diminished the FoxO (forkhead box O) 3a signaling cascade to exert protective effects of MYDGF on LDL transcytosis and atherosclerosis. The findings support a role for inflammatory cell-derived MYDGF served as a cross talk factor between inflammatory cells and endothelial cells that inhibits LDL transcytosis across endothelium. MYDGF may become a novel therapeutic drug for atherosclerosis, and the beneficial effects of inflammatory cell in atherosclerosis deserve further attention.

Effect of Ligustrazine on Lipopolysaccharide-induced Inflammatory Response of Vascular Endothelial Cells and its Possible Mechanism

Background Endothelial cell (EC) inflammation plays a crucial role in the development of several cardiovascular disorders (CD), including atherosclerosis and sepsis. Ligustrazine (Lig), a bioactive constituent derived from Traditional Chinese Medicine Ligusticum chuanxiong Hort, has exhibited in vivo anti-inflammatory properties. Despite the observed positive outcomes, the exact mechanisms underlying these beneficial effects remain unidentified. Aim The goal of this research is to investigate the influence and potential mechanism of Lig on lipopolysaccharide (LPS)-induced inflammatory responses in human umbilical vein endothelial cells (HUVECs). Introduction These experiments investigate the effectiveness of Lig in preventing LPS-induced damage in HUVECs, with the goal of elucidating the underlying processes at work. Materials and Methods To evaluate HUVECs’ viability, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was conducted. Enzyme-linked immunosorbent assay (ELISA) was employed to measure changes in ICAM-1, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein (MCP-1) levels. Real-time polymerase chain reaction (RT-PCR) was used to determine the levels of ICAM-1, IL-6, TNF-α, MCP-1, and toll-like receptors (TLR4) mRNA. Additionally, we performed WB analysis to assess the levels of nuclear factor-κB (NF-κB) p65, TLR4, IκBα, and p-IκBα. Results The findings demonstrated significantly suppressed cell viability due to LPS treatment, while Lig treatment increased cell viability in a concentration-dependent manner. Lig also effectively reduced the mRNA levels of ICAM-1, TNF-α, IL-6, and MCP-1. Furthermore, Lig pretreatment led to downregulation of TLR4, p-IκBα, and NF-κB p65 in HUVECs. Discussion The findings indicate that Lig reduces LPS-induced inflammation in HUVECs, and that the TLR4/NF-κB pathway is critical in increasing cell survival and minimizing inflammatory damage. This provides possible anti-inflammatory techniques for treating CD. Conclusion In conclusion, our work demonstrates Lig’s anti-inflammatory actions on LPS-stimulated HUVECs. The data suggest that Lig lowers inflammation via regulating the TLR4/NF-B pathway, boosting cell survival, and decreasing inflammatory responses.

Roles of Protein S-Nitrosylation in Endothelial Homeostasis and Dysfunction.

Significance: Nitric oxide (NO) plays several distinct roles in endothelial homeostasis. Except for activating the guanylyl cyclase enzyme-dependent cyclic guanosine monophosphate signaling pathway, NO can bind reactive cysteine residues in target proteins, a process known as S-nitrosylation (SNO). SNO is proposed to explain the multiple biological functions of NO in the endothelium. Investigating the targets and mechanism of protein SNO in endothelial cells (ECs) can provide new strategies for treating endothelial dysfunction-related diseases. Recent Advances: In response to different environments, proteomics has identified multiple SNO targets in ECs. Functional studies confirm that SNO regulates NO bioavailability, inflammation, permeability, oxidative stress, mitochondrial function, and insulin sensitivity in ECs. It also influences EC proliferation, migration, apoptosis, and transdifferentiation. Critical Issues: Single-cell transcriptomic analysis of ECs isolated from different mouse tissues showed heterogeneous gene signatures. However, litter research focuses on the heterogeneous properties of SNO proteins in ECs derived from different tissues. Although metabolism reprogramming plays a vital role in endothelial functions, little is known about how protein SNO regulates metabolism reprogramming in ECs. Future Directions: Precisely deciphering the effects of protein SNO in ECs isolated from different tissues under different conditions is necessary to further characterize the relationship between protein SNO and endothelial dysfunction-related diseases. In addition, identifying SNO targets that can influence endothelial metabolic reprogramming and the underlying mechanism can offer new views on the crosstalk between metabolism and post-translational protein modification. Antioxid. Redox Signal. 40, 186-205.

GSDME in Endothelial Cells: Inducing Vascular Inflammation and Atherosclerosis via Mitochondrial Damage and STING Pathway Activation.

The initiation of atherosclerotic plaque is characterized by endothelial cell inflammation. In light of gasdermin E's (GSDME) role in pyroptosis and inflammation, this study elucidates its function in atherosclerosis onset. Employing Gsdme- and apolipoprotein E-deficient (Gsdme-/-/ApoE-/-) and ApoE-/- mice, an atherosclerosis model was created on a Western diet (WD). In vitro examinations with human umbilical vein endothelial cells (HUVECs) included oxidized low-density lipoprotein (ox-LDL) exposure. To explore the downstream mechanisms linked to GSDME, we utilized an agonist targeting the stimulator of the interferon genes (STING) pathway. The results showed significant GSDME activation in ApoE-/- mice arterial tissues, corresponding with atherogenesis. Gsdme-/-/ApoE-/- mice displayed fewer plaques and decreased vascular inflammation. Meanwhile, GSDME's presence was confirmed in endothelial cells. GSDME inhibition reduced the endothelial inflammation induced by ox-LDL. GSDME was linked to mitochondrial damage in endothelial cells, leading to an increase in cytoplasmic double-stranded DNA (dsDNA). Notably, STING activation partially offset the effects of GSDME inhibition in both in vivo and in vitro settings. Our findings underscore the pivotal role of GSDME in endothelial cells during atherogenesis and vascular inflammation, highlighting its influence on mitochondrial damage and the STING pathway, suggesting a potential therapeutic target for vascular pathologies.

Pelargonidin alleviates acrolein-induced inflammation in human umbilical vein endothelial cells by reducing COX-2 expression through the NF-κB pathway.

Acrolein, a common environmental pollutant, is linked to the development of cardiovascular inflammatory diseases. Pelargonidin is a natural compound with anti-inflammation activity. In this study, we aimed to explore the effects of pelargonidin on inflammation induced by acrolein in human umbilical vein endothelial cells (HUVECs). MTT assay was utilized for assessing cell viability in HUVECs. LDH release in HUVECs was measured using the LDH kit. Western blot was used to detect the protein expression of p-p65, p65 and COX-2. Inflammation was evaluated through determining the levels of PGE2, IL-1β, IL-6, IL-8 and TNF-α in HUVECs after treatment. COX-2 mRNA expression and COX-2 content were examined using RT-qPCR and a human COX-2 ELISA kit, respectively. Acrolein treatment at 50μM resulted in a 45% decrease in the viability and an increase in LDH release (2.2-fold) in HUVECs. Pelargonidin at 5, 10, 20, and 40μM alleviated acrolein-caused inhibitory effect on cell viability (increased to 1.3-, 1.5-, 1.8-, and 1.9-fold, respectively, compared to acrolein treatment group) and promoting effect on LDH release (decreased to 82%, 75%, 62%, and 58%, respectively, compared to acrolein treatment group) in HUVECs. Moreover, pelargonidin or pyrrolidine dithiocarbamate (PDTC; an NF-κB pathway inhibitor) inhibited acrolein-induced activation of the NF-κB pathway. Acrolein elevated the levels of PGE2, IL-1β, IL-6, IL-8 and TNF-α (from 40.2, 27.3, 67.2, 29.0, 24.8pg/mL in control group to 224.0, 167.3, 618.3, 104.6, and 275.1pg/mL in acrolein treatment group, respectively), which were retarded after pelargonidin (decreased to 134.8, 82.3, 246.2, 70.2, and 120.8pg/mL in acrolein + pelargonidin treatment group) or PDTC (decreased to 107.9, 80.1, 214.6, 64.0, and 96.6pg/mL in acrolein + PDTC treatment group) treatment in HUVECs. Pelargonidin inactivated the NF-κB pathway to reduce acrolein-induced COX-2 expression. Furthermore, pelargonidin relieved acrolein-triggered inflammation through decreasing COX-2 expression by inactivating the NF-κB pathway in HUVECs. In conclusion, pelargonidin could protect against acrolein-triggered inflammation in HUVECs through attenuating COX-2 expression by inactivating the NF-κB pathway.

Amphipathic Helical Peptide L37pA Protects against Lung Vascular Endothelial Dysfunction Caused by Truncated Oxidized Phospholipids via Antagonism with CD36 Receptor.

The generation of bioactive truncated oxidized phospholipids (Tr-OxPLs) from oxidation of cell-membrane or circulating lipoproteins is a common feature of various pathological states. Scavenger receptor CD36 is involved in lipid transport and acts as a receptor for Tr-OxPLs. Interestingly, Tr-OxPLs and CD36 are involved in endothelial dysfunction-derived acute lung injury, but the precise mechanistic connections remain unexplored. In the present study, we investigated the role of CD36 in mediating pulmonary endothelial cell (EC) dysfunction caused by Tr-OxPLs. Our results demonstrated that the Tr-OxPLs KOdia-PC, Paz-PC, PGPC, PON-PC, POV-PC, and lysophosphocholine caused an acute EC barrier disruption as revealed by measurements of transendothelial electrical resistance and VE-cadherin immunostaining. More importantly, a synthetic amphipathic helical peptide, L37pA, targeting human CD36 strongly attenuated Tr-OxPL-induced EC permeability. L37pA also suppressed Tr-OxPL-induced endothelial inflammatory activation monitored by mRNA expression of inflammatory cytokines/chemokines and adhesion molecules. In addition, L37pA blocked Tr-OxPL-induced NF-κB activation and tyrosine phosphorylation of Src kinase and VE-cadherin. The Src inhibitor SU6656 attenuated KOdia-PC-induced EC permeability and inflammation, but inhibition of the Toll-like receptors (TLRs) TLR1, TLR2, TLR4, and TLR6 had no such protective effects. CD36-knockout mice were more resistant to Tr-OxPL-induced lung injury. Treatment with L37pA was equally effective in ameliorating Tr-OxPL-induced vascular leak and lung inflammation as determined by an Evans blue extravasation assay and total cell and protein content in BAL fluid. Altogether, these results demonstrate an essential role of CD36 in mediating Tr-OxPL-induced EC dysfunction and suggest a strong therapeutic potential of CD36 inhibitory peptides in mitigating lung injury and inflammation.