Endothelial Proinflammatory Responses Research Articles

The Effects of the Levosimendan Metabolites OR-1855 and OR-1896 on Endothelial Pro-Inflammatory Responses

The calcium sensitizer levosimendan is used for the treatment of acute decompensated heart failure. A small portion (4-7%) of levosimendan is metabolized to the pharmacologically active metabolite OR-1896 via the inactive intermediate OR-1855. In addition, levosimendan has been shown to exert positive effects on the endothelium in vitro antagonizing vascular dysfunction and inflammation. However, the function of the levosimendan metabolites within this context is still unknown. In this study, we thus investigated the impact of the metabolites OR-1896 and OR-1855 on endothelial inflammatory processes in vitro. We observed a reduction of IL-1β-dependent endothelial adhesion molecule ICAM-1 and VCAM-1 as well as interleukin (IL) -6 expression upon levosimendan treatment but not after treatment with OR-1855 or OR-1896, as assessed by western blotting, flow cytometry, and qRT-PCR. Instead, the metabolites impaired IL-1β-induced ROS formation via inactivation of the MAPK p38, ERK1/2, and JNK. Our results suggest that the levosimendan metabolites OR-1896 and OR-1855 have certain anti-inflammatory properties, partly other than levosimendan. Importantly, they additionally show that the intermediate metabolite OR-1855 does, in fact, have pharmacological effects in the endothelium. This is interesting, as the metabolites are responsible for the long-term therapeutic effects of levosimendan, and heart failure is associated with vascular dysfunction and inflammation.

Disturbed Flow-Facilitated Margination and Targeting of Nanodisks Protect against Atherosclerosis.

Disturbed blood flow induces endothelial pro-inflammatory responses that promote atherogenesis. Nanoparticle-based therapeutics aimed at treating endothelial inflammation in vasculature where disturbed flow occurs may provide a promising avenue to prevent atherosclerosis. By using a vertical-step flow apparatus and a microfluidic chip of vascular stenosis, herein, it is found that the disk-shaped versus the spherical nanoparticles exhibit preferential margination (localization and adhesion) to the regions with the pro-atherogenic disturbed flow. By employing a mouse model of carotid partial ligation, superior targeting and higher accumulation of the disk-shaped particles are also demonstrated within disturbed flow areas than that of the spherical particles. In hyperlipidemia mice, administration of disk-shaped particles loaded with hypomethylating agent decitabine (DAC) displays greater anti-inflammatory and anti-atherosclerotic effects compared with that of the spherical counterparts and exhibits reduced toxicity than "naked" DAC. The findings suggest that shaping nanoparticles to disk is an effective strategy for promoting their delivery to atheroprone endothelia.

Endothelial UCP2 Is a Mechanosensitive Suppressor of Atherosclerosis.

Background:Inflamed endothelial cells (ECs) trigger atherogenesis, especially at arterial regions experiencing disturbed blood flow. UCP2 (Uncoupling protein 2), a key mitochondrial antioxidant protein, improves endothelium-dependent relaxation in obese mice. However, whether UCP2 can be regulated by shear flow is unknown, and the role of endothelial UCP2 in regulating inflammation and atherosclerosis remains unclear. This study aims to investigate the mechanoregulation of UCP2 expression in ECs and the effect of UCP2 on endothelial inflammation and atherogenesis.Methods:In vitro shear stress simulation system was used to investigate the regulation of UCP2 expression by shear flow. EC-specific Ucp2 knockout mice were used to investigate the role of UCP2 in flow-associated atherosclerosis.Results:Shear stress experiments showed that KLF2 (Krüppel-like factor 2) mediates fluid shear stress-dependent regulation of UCP2 expression in human aortic and human umbilical vein ECs. Unidirectional shear stress, statins, and resveratrol upregulate whereas oscillatory shear stress and proinflammatory stimuli inhibit UCP2 expression through altered KLF2 expression. KLF2 directly binds to UCP2 promoter to upregulate its transcription in human umbilical vein ECs. UCP2 knockdown induced expression of genes involved in proinflammatory and profibrotic signaling, resulting in a proatherogenic endothelial phenotype. EC-specific Ucp2 deletion promotes atherogenesis and collagen production. Additionally, we found endothelial Ucp2 deficiency aggravates whereas adeno-associated virus-mediated EC-Ucp2 overexpression inhibits carotid atherosclerotic plaque formation in disturbed flow-enhanced atherosclerosis mouse model. RNA-sequencing analysis revealed FoxO1 (forkhead box protein O1) as the major proinflammatory transcriptional regulator activated by UCP2 knockdown, and FoxO1 inhibition reduced vascular inflammation and disturbed flow-enhanced atherosclerosis. We showed further that UCP2 level is critical for phosphorylation of AMPK (AMP-activated protein kinase), which is required for UCP2-induced inhibition of FoxO1.Conclusions:Altogether, our studies uncover that UCP2 is novel mechanosensitive gene under the control of fluid shear stress and KLF2 in ECs. UCP2 expression is critical for endothelial proinflammatory response and atherogenesis. Therapeutic strategies enhancing UCP2 level may have therapeutic potential against atherosclerosis.

Internalization of cell-derived microparticles triggers endothelial pro-inflammatory responses.

Increased numbers of circulating microparticles (MPs) have long been documented in thalassemia and are considered as a contributing factor in developing the thromboembolic events (TEEs), which are associated with endothelial dysfunction. Indeed, the cellular and molecular mechanisms by which MPs and endothelial cells interact and their consequences remain poorly investigated. The present study aims to compare the biological effects of MPs obtained from healthy subjects and β-thalassemia/HbE patients on endothelial pro-inflammatory responses. MPs isolated from plasma by two-step centrifugation from 10 healthy donors, 19 splenectomized and 30 non-splenectomized β-thalassemia/HbE patients were first characterized for their cellular origins, then counted and incubated with primary human umbilical vein endothelial cells (HUVECs). Internalization of MPs into HUVECs and their induction on endothelial cell activation and pro-inflammatory responses were determined. MPs either from healthy or β-thalassemia/HbE patients could become internalized into endothelial cells, but unlike MPs from healthy donors and non-splenectomized patients, MPs from splenectomized patients were the most active and induced the 2-fold up-regulation of pro-inflammatory genes, IL1B, CXCL8, and CCL2 and 4-fold increase in interleukin-1β. In addition, MPs from both healthy subjects and splenectomized patients at 106/ml failed to trigger the secretion of endothelial IL-6 and IL-8 while higher MP concentration at 5 × 10⁶/ml significantly induced this secretion. Plasma MPs isolated from splenectomized β-thalassemia/HbE patients are capable of triggering pro-inflammatory responses from endothelial cells reflected at both gene and protein levels.

Metformin ameliorates endotoxemia-induced endothelial pro-inflammatory responses via AMPK-dependent mediation of HDAC5 and KLF2

Exaggerated endothelial pro-inflammatory response is a hallmark in the early stage of sepsis and contributes to the subsequent tissue injury and organ failure. The anti-inflammatory effects of AMP-activated protein kinase (AMPK) activator metformin in sepsis has been revealed. However, the underlying mechanisms remain not fully understood. In the present study, the potential roles of histone deacetylase 5 (HDAC5) and kruppel-like factor 2 (KLF2) in the effects of metformin on endothelial pro-inflammatory responses were investigated. The results showed that metformin pretreatment increased the phosphorylation of HDAC5 at serine 498, leading to the upregulation of KLF2, and eliminated lipopolysaccharide (LPS) and tumor necrosis factor ⍺ (TNF⍺)-induced upregulation of vascular cell adhesion molecule 1 (VCAM1). Furthermore, the adhesion of HL60 leukocytes to endothelial monolayer was effectively inhibited by metformin. In addition, the in vivo data confirmed that AMPK activation attenuated local and systemic inflammation in endotoxic mice induced by LPS via mediating phosphorylating HDAC5 and restoring KLF2 expression. Our findings revealed that AMPK activation-mediated HDAC5 phosphorylation and KLF2 restoration is, at least partially, responsible to the anti-inflammatory effects of metformin in endotoxemia-induced endothelial cells, which has important implications for the future development of interfering therapies of sepsis.

Atypical Gasdermin D and Mixed Lineage Kinase Domain-like Protein Leakage Aggravates Tetrachlorobenzoquinone-Induced Nod-like Receptor Protein 3 Inflammasome Activation.

Our previous study showed that tetrachlorobenzoquinone (TCBQ) mediated the activation of Nod-like receptor protein 3 (NLRP3) inflammasome, which involves K+ efflux, reactive oxygen species (ROS) production, and mitochondrial DNA damage. In addition, TCBQ down-regulates NLRP3 ubiquitination and promotes the activation of NLRP3 inflammasome. However, the induction of NLRP3 inflammasome by atypical pathways has not yet been characterized. Using human umbilical vein endothelial cells (HUVEC), we discovered that TCBQ activates caspase 1/4/5 and cleaves gasdermin D (GSDMD) into N-terminal and C-terminal cleavage products. In parallel, TCBQ also activates receptor interacting protein kinase 3 (RIPK3)/mixed lineage kinase domain-like protein (MLKL) signaling pathways. The N-terminal fragments of GSDMD and MLKL translocate from cytoplasm to cell membrane and form oligomers and membrane pores on the cell membrane. The formation of membrane pores not only promotes the extracellular secretion of interleukin 1 beta (IL-1β) but also affects cellular ion homeostasis, in particular promotes K+ outflow, which further activates NLRP3 inflammasome and aggravates cellular inflammation. These results indicated that GSDMD and MLKL play important roles in TCBQ-induced endothelial pro-inflammatory responses, which may point to potential therapeutic approaches for TCBQ-mediated toxicity.

The Glycolytic Enzyme PFKFB3 Controls TNF-α-Induced Endothelial Proinflammatory Responses.

Endothelial cells play an important role in health and a variety of diseases. Recent evidences show that endothelial cells rely on glycolysis rather than on oxidative phosphorylation to generate energy to support cellular functions such as angiogenesis. However, the effect of endothelial glycolysis on vascular inflammation remains little known. Here, we investigate the role of key glycolytic enzyme PFKFB3 in tumor necrosis factor-α (TNF-α)-induced endothelial proinflammatory responses. siRNAs were used to knockdown the expression of PFKFB3. In some experiments, PFKFB3 inhibitors were also used. TNF-α at 20ng/ml was added to confluent endothelial cells for different time period of stimulation. PFKFB3 expression was examined by RT-PCR and western blotting. Cytokine antibody panel membranes were employed to detect different cytokines/chemokines in culture supernatant of endothelial cells. The determination of monocyte adhesion to endothelial cells after TNF-α treatment was conducted using THP-1 cells. The monocyte attraction was performed using Transwell filters. For further mechanisms, NF-κB-p65 localization was examined by immunofluorescence. Expression of total IκB, phospho-IκB, phospho-NF-κB-p65, and Ikkβ was detected by western blotting. DNA-binding activity of NF-κB was assessed using electrophoretic mobility shift assay. We found that TNF-α increased endothelial PFKFB3 expression. Knockdown of PFKFB3 almost blocked all TNF-α-induced release of the proinflammatory cytokines/chemokines (MCP-1, IL-8, CXCL1, GMCSF, RANTES, TNF-α) and ICAM-1. PFKFB3 knockdown also significantly inhibited TNF-α-induced monocyte adhesion and transmigration. Furthermore, inhibition of PFKFB3 inhibited TNF-α-induced Ikkβ phosphorylation, IκBα phosphorylation and degradation, NF-κB-p65 phosphorylation, nuclear translocation, and DNA-binding activity. Thus, our results demonstrate that glycolytic enzyme PFKFB3 plays a critical role in TNF-α-induced endothelial inflammation.

Rosuvastatin Inhibits Interleukin (IL)-8 and IL-6 Production in Human Coronary Artery Endothelial Cells Stimulated With Aggregatibacter actinomycetemcomitans Serotype b.

Rosuvastatin exhibits anti-inflammatory effects and reduces periodontal diseases and atherosclerosis; however, its role in regulating periodontopathogen-induced endothelial proinflammatory responses remains unclear. The purpose of this study is to determine whether rosuvastatin can reduce the proinflammatory response induced by Aggregatibacter actinomycetemcomitans (Aa) in human coronary artery endothelial cells (HCAECs). HCAECs were stimulated with purified Aa serotype b lipopolysaccharide (LPS) (Aa-LPS), heat-killed (HK) bacteria (Aa-HK), or live bacteria. Expression of Toll-like receptors and cellular adhesion molecules were evaluated by fluorometric enzyme-linked immunosorbent assay. Endothelial cell activation was evaluated by quantifying nuclear factor (NF)-kappa B-p65 and cytokine expression levels by quantitative polymerase chain reaction and flow cytometry. Effect of rosuvastatin in expression of the atheroprotective factor Krüppel-like factor 2 (KLF2) and cytokines were also studied using similar approaches. HCAECs showed increased interleukin (IL)-6, IL-8, intercellular adhesion molecule 1, and platelet endothelial cell adhesion molecule 1 expression when stimulated with Aa-LPS or Aa-HK. NF-κB-p65 activation was induced by all antigens. Aa-induced IL-6 and IL-8 production was inhibited by rosuvastatin, particularly at higher doses. Interestingly, reduced IL-6 and IL-8 levels were observed in HCAECs stimulated with Aa in the presence of higher concentrations of rosuvastatin. This anti-inflammatory effect correlated with a significant increase of rosuvastatin-induced KLF2. These results suggest Aa-induced proinflammatory endothelial responses are regulated by rosuvastatin in a mechanism that appears to involve KLF2 activation. Use of rosuvastatin to prevent cardiovascular disease may reduce risk of endothelial activation by bacterial antigens.

Parkin Mediates Endothelial Pro-Inflammatory Responses in Acute Lung Injury

Parkin Mediates Endothelial Pro-Inflammatory Responses in Acute Lung Injury

Histone Deacetylase Inhibition and IκB Kinase/Nuclear Factor-κB Blockade Ameliorate Microvascular Proinflammatory Responses Associated With Hemorrhagic Shock/Resuscitation in Mice.

To investigate the consequences of histone deacetylase inhibition by histone deacetylase inhibitor valproic acid and IκB kinase/nuclear factor-κB signaling blockade by IκB kinase inhibitor BAY11-7082 on (microvascular) endothelial cell behavior in vitro as well as in mice subjected to hemorrhagic shock/resuscitation in vivo. Prospective, randomized laboratory investigation using an established mouse model of hemorrhagic shock. Research laboratory at university teaching hospital. Endothelial cells and C57BL/6 male mice. Endothelial cells were incubated with tumor necrosis factor-α in the absence or presence of valproic acid or BAY11-7082 in vitro. Mice were subjected to hemorrhagic shock by blood withdrawn until the mean arterial pressure of 30 mm Hg and maintained at this pressure for 90 minutes. At 90 minutes, subgroups of mice were resuscitated with 4% human albumin in the absence or presence of vehicle, valproic acid (300 μg/g body weight) or BAY11-7082 (400 μg per mouse). Mice were killed 1 hour and 4 hours after resuscitation. Valproic acid and BAY11-7082 selectively diminished tumor necrosis factor-α-induced endothelial proinflammatory activation in vitro. In vivo, both systemic and local inflammatory responses were significantly induced by hemorrhagic shock/resuscitation. The decreased histone acetylation in kidneys after hemorrhagic shock/resuscitation was restored by valproic acid treatment. In glomerular endothelial cells, the nuclear translocation of nuclear factor-κB, which was induced by hemorrhagic shock/resuscitation, was eliminated by BAY11-7082 treatment while enhanced in the presence of valproic acid. Both valproic acid and BAY11-7082 significantly attenuated the hemorrhagic shock/resuscitation-induced protein expression of endothelial cell adhesion molecules E-selectin and vascular cell adhesion molecule-1 in the microvasculature of kidneys and liver, although messenger RNA expression levels of these molecules analyzed in whole-organ lysates of kidneys, lungs, and liver were not extensively affected. The reduced protein expression of adhesion molecules was paralleled by diminishing the adhesion/transmigration of neutrophils in kidneys and liver after hemorrhagic shock/resuscitation. Suppression of histone deacetylase activity and blockade of IκB kinase/nuclear factor-κB signaling during resuscitation ameliorate microvascular endothelial proinflammatory responses in organs in mice after hemorrhagic shock.

Importance of endothelial NF-κB signalling in vascular remodelling and aortic aneurysm formation

Vascular remodelling and aortic aneurysm formation are induced mainly by inflammatory responses in the adventitia and media. However, relatively little is known about the mechanistic significance of endothelium in the pathogenesis of these vascular disorders. The transcription factor nuclear factor-kappa B (NF-κB) regulates the expressions of numerous genes, including those related to pro-inflammatory responses. Therefore, to investigate the roles of endothelial pro-inflammatory responses, we examined the impact of blocking endothelial NF-κB signalling on intimal hyperplasia and aneurysm formation. To block endothelial NF-κB signalling, we used transgenic mice expressing dominant-negative IκBα selectively in endothelial cells (E-DNIκB mice). E-DNIκB mice were protected from the development of cuff injury-induced neointimal formation, in association with suppressed arterial expressions of cellular adhesion molecules, a macrophage marker, and inflammatory factors. In addition, the blockade of endothelial NF-κB signalling prevented abdominal aortic aneurysm formation in an experimental model, hypercholesterolaemic apolipoprotein E-deficient mice with angiotensin II infusion. In this aneurysm model as well, aortic expressions of an adhesion molecule, a macrophage marker, and inflammatory factors were suppressed with the inhibited expression and activity of matrix metalloproteinases in the aorta. Endothelial NF-κB activation up-regulates adhesion molecule expression, which may trigger macrophage infiltration and inflammation in the adventitia and media. Thus, the endothelium plays important roles in vascular remodelling and aneurysm formation through its intracellular NF-κB signalling.

Endothelial cell activation by antiphospholipid antibodies is modulated by Krüppel-like transcription factors

AbstractAntiphospholipid syndrome is characterized by thrombosis and/or recurrent pregnancy loss in the presence of antiphospholipid antibodies (APLAs). The majority of APLAs are directed against phospholipid-binding proteins, particularly β2-glycoprotein I (β2GPI). Anti-β2GPI antibodies activate endothelial cells in a β2GPI-dependent manner through a pathway that involves NF-κB. Krüppel-like factors (KLFs) play a critical role in regulating the endothelial response to inflammatory stimuli. We hypothesized that activation of endothelial cells by APLA/anti-β2GPI antibodies might be associated with decreased expression of KLFs, which in turn might facilitate cellular activation mediated through NF-κB. Our experimental results confirmed this hypothesis, demonstrating markedly decreased expression of KLF2 and KLF4 after incubation of cells with APLA/anti-β2GPI antibodies. Restoration of KLF2 or KLF4 levels inhibited NF-κB transcriptional activity and blocked APLA/anti-β2GPI–mediated endothelial activation despite NF-κB p65 phosphorylation. Chromatin immunoprecipitation analysis demonstrated that inhibition of NF-κB transcriptional activity by KLFs reflects sequestration of the cotranscriptional activator CBP/p300, making this cofactor unavailable to NF-κB. These findings suggest that the endothelial response to APLA/anti-β2GPI antibodies reflects competition between KLFs and NF-κB for their common cofactor, CBP/p300. Taken together, these observations are the first to implicate the KLFs as novel participants in the endothelial proinflammatory response to APLA/anti-β2GPI antibodies.

Gamma Interferon Confers Resistance to Infection withStaphylococcus aureusin Human Vascular Endothelial Cells by Cooperative Proinflammatory and Enhanced Intrinsic Antibacterial Activities

Vascular endothelium is an exposed target in systemic endovascular Staphylococcus aureus infections. We reported earlier that the proinflammatory and procoagulant activities of primary human umbilical vein endothelial cells (ECs) after binding and ingestion of S. aureus organisms provide the cells effective means for leukocyte-mediated bacterial elimination. Expanding on this, we now show that these ECs exhibit a modest intrinsic capacity for eliminating intracellular S. aureus that was influenced by cytokines relevant to S. aureus infections. Using various EC infection assays, we showed that gamma interferon (IFN-gamma), applied to cultures of ECs prior to or after infection with S. aureus, markedly reduced the level of infection, illustrated by lower percentages of S. aureus-infected ECs and less intracellular bacteria per infected cell. IFN-gamma-activated ECs had unaltered abilities to bind S. aureus and processed ingested bacteria by a seemingly conventional phagocytic pathway. IFN-gamma treatment rescued EC monolayers from severe injury by virulent clinical S. aureus strains or excessive bacterial numbers. Mechanistically, IFN-gamma controls S. aureus infection via IFN-gamma receptor, most likely through stimulation of intrinsic endothelial antibacterial mechanisms but independent of processes that deprive bacteria of intracellular L-tryptophan or iron. The antibacterial activity of IFN-gamma-stimulated ECs coincided with sustained or slightly elevated endothelial proinflammatory responses that supported monocyte recruitment. In conclusion, we identify IFN-gamma as a potent regulatory Th1 cytokine possessing exclusive abilities to augment intrinsic antistaphylococcal effector mechanisms in human ECs without ablating the S. aureus-induced proinflammatory EC responses and, as such, coordinating a protective efficacy of ECs against blood-borne S. aureus infection.

Glutathione Peroxidase Mimics Prevent TNFα- and Neutrophil-Induced Endothelial Alterations

Based on the assumption that glutathione peroxidase (GPx) activity might be limiting in preventing peroxide-induced impairment of endothelial regulatory functions, we studied the effect of a series of new selenium-containing GPx mimics on endothelial cells exposed to an inflammatory stress. The two compounds that have the highest GPx activity, BXT-51072 and BXT-51077, were shown to be the most efficient inhibitors of leukocyte recruitment by human umbilical vein endothelial cells (HUVEC), upon incubation with neutrophils (10-fold excess over HUVEC) and with 1 ng/ml TNF-α for 1 or 3.5 h. When HUVEC were pre- and cotreated with 10 μM of either compound, neutrophil adhesion and endothelial alteration were markedly inhibited, as assessed by immunoassays of myeloperoxidase and von Willebrand factor, respectively. These two GPx mimics were also found to be the most efficient inhibitors of the TNFα-induced endothelial expression of P- and E-selectin and of the TNFα- or interleukin1-induced endothelial release of interleukin-8. Our results demonstrate that GPx mimics such as BXT-51072 behave as potent antagonists of TNF-α and interleukin-1 through the downregulation of endothelial proinflammatory responses.