THP-1 Monocyte Adhesion Research Articles

Differential Effects of Ruminant and Industrial 18-Carbon trans-Monounsaturated Fatty Acids (trans Vaccenic and Elaidic) on the Inflammatory Responses of an Endothelial Cell Line.

Endothelial dysfunction and inflammation are recognised factors in the development of atherosclerosis. Evidence suggests that intake of industrial trans fatty acids (TFAs) promotes endothelial dysfunction, while ruminant TFAs may have the opposite effect. The aim of this study was to compare the effects of elaidic acid (EA (18:1n-9t); an industrially produced TFA) and trans vaccenic acid (TVA (18:1n-7t); a natural TFA found in ruminant milk and meat) on inflammatory responses of endothelial cells (ECs). ECs (EA.hy926 cells) were cultured under standard conditions and exposed to TFAs (1 to 50 μM) for 48 h. Then, the cells were cultured for a further 6 or 24 h with tumour necrosis factor alpha (TNF-α, 1 ng/mL) as an inflammatory stimulant. ECs remained viable after treatments. TFAs were incorporated into ECs in a dose-dependent manner. Preincubation with EA (50 µM) increased production of MCP-1, RANTES, and IL-8 in response to TNF-α, while preincubation with TVA (1 µM) decreased production of ICAM-1 and RANTES in response to TNF-α. Preincubation with EA (50 µM) upregulated toll-like receptor 4 and cyclooxygenase 2 gene expression in response to TNF-α. In contrast, preincubation with TVA (1 µM) downregulated TNF-α induced nuclear factor kappa B subunit 1 gene expression. Preincubation of ECs with EA (50 µM) increased THP-1 monocyte adhesion. In contrast, preincubation of ECs with TVA (1 µM) reduced THP-1 monocyte adhesion, while preincubation of ECs with TVA (50 µM) decreased the level of surface expression of ICAM-1 seen following TNF-α stimulation. The results suggest that TVA has some anti-inflammatory properties, while EA enhances the response to an inflammatory stimulus. These findings suggest differential effects induced by the TFAs tested, fitting with the idea that industrial TFAs and ruminant TFAs can have different and perhaps opposing biological actions in an inflammatory context.

Tomatidine Improves Pulmonary Inflammation in Mice with Acute Lung Injury.

Tomatidine, which is isolated from green tomato, can ameliorate inflammation and oxidative stress in cells and animal experiments and has been shown to improve airway inflammation in a murine model of asthma. Here, we investigated whether tomatidine can ameliorate acute lung injury in mice. Mice were given tomatidine by intraperitoneal injection for 7 consecutive days, and then, lung injury was induced via intratracheal instillation of lipopolysaccharide (LPS). Tomatidine reduced inflammatory cytokine expressions in bronchoalveolar lavage fluid (BALF), attenuated neutrophil infiltration in the BALF and lung tissue, increased superoxide dismutase activity and glutathione levels, and alleviated myeloperoxidase expression in the lung tissue of mice with lung injury. Tomatidine also decreased inflammatory cytokine and chemokine gene expression in inflammatory lungs and attenuated the phosphorylation of mitogen-activated protein kinase and nuclear factor kappa B. Furthermore, tomatidine enhanced the production of heme oxygenase-1, decreased the secretion of inflammatory cytokines and chemokines in LPS-stimulated lung epithelial cells, and attenuated THP-1 monocyte adhesion. Our findings suggest that tomatidine attenuates oxidative stress and inflammation, improving acute lung injury in mice.

Abstract P260: SARS-Cov-2 Spike Protein S1-Mediated Endothelial Injury And Pro-Inflammatory State Is Amplified By Dihydrotestosterone And Prevented By Mineralocorticoid Antagonism

Objectives: Men are disproportionately affected by the coronavirus disease-2019 (COVID-19) and experience higher mortality as compared to women. Endothelial dysfunction has been proposed as a major inciting factor of pro-inflammatory and thrombotic changes in COVID-19 infection. The vascular effects of androgen signaling and inflammatory cytokines in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mediated endothelial damage are not defined. Methods: Here, we determined the effects of SARS-CoV-2 spike protein-mediated endothelial damage under conditions of exposure to androgens and TNF-α. We tested the therapeutic effects of approved drugs: angiotensin receptor blockade by valsartan and mineralocorticoid receptor antagonism by spironolactone. Circulating endothelial injury markers VCAM-1 and E-selectin were measured in the plasma from men and women diagnosed with COVID-19. Results: Exposure of endothelial cells (ECs) in vitro to androgen dihydrotestosterone (DHT) exacerbated spike protein S1-mediated endothelial injury transcripts for cell adhesion molecules E-selectin, VCAM-1, and ICAM-1 and anti-fibrinolytic PAI-1 (P<0.05), as well as increased THP-1 monocyte adhesion to ECs (P=0.032). Spironolactone dramatically reduced DHT+S1-induced endothelial activation. Pre-treatment of ECs with valsartan did not increase endothelial activation by S1. TNF-α exacerbated S1-induced EC activation and spironolactone abrogated this effect. Analysis of plasma from patients hospitalized with COVID-19 showed concordant higher VCAM-1 in men as compared to women. Conclusions: Androgen exposure promoted spike protein-mediated endothelial injury by increasing markers of inflammation and thrombosis. A beneficial therapeutic effect of the FDA-approved drug spironolactone was observed, supporting a rationale for further evaluation as an adjunct treatment in COVID-19.

P2Y12 receptor blockers are anti-inflammatory drugs inhibiting both circulating monocytes and macrophages including THP-1 cells

P2Y12 blockade improves patient outcomes after myocardial infarction. As well as antithrombotic effects, anti-inflammatory effects may contribute to this beneficial clinical outcome. Here we aimed to identify potential anti-inflammatory effects of P2Y12 receptor blockers on monocytes and macrophages. Using flow cytometry, migration assays, flow chambers and RNA microarrays, we investigated the effects of adenosine diphosphate (ADP) and P2Y12 receptor blockers on blood monocytes, THP-1 monocytes and THP-1 monocytes after differentiation to macrophages. P2Y12 -expressing platelets can form aggregates with monocytes in circulating blood. Mediated by platelets, ADP results in activation of the integrin receptor Mac-1 on blood monocytes, as detected by the conformation-specific single-chain antibody MAN-1. Via the same association with platelets, THP-1 monocyte adhesion to the endothelial intercellular adhesion molecule 1 (ICAM-1) is induced by ADP. P2Y12 receptor blockers prevent these ADP effects on monocytes. Interestingly, in contrast to THP-1 monocytes, THP-1 monocytes, after differentiation to macrophages, directly expressed the P2Y12 receptor and consequently ADP was found to be a potent chemoattractant. Again, P2Y12 receptor blockers antagonised this effect. Accordingly, stimulation of THP-1 macrophages with ADP caused a substantial change in gene expression pattern and upregulation of several genes associated with inflammation and atherogenesis. These data establish novel anti-inflammatory effects of P2Y12 receptor blockers on monocytes and macrophages, which are expected to contribute to cardiovascular risk reduction.

Activation of GPR30 with G1 inhibits oscillatory shear stress-induced adhesion of THP-1 monocytes to HAECs by increasing KLF2.

Atherosclerosis is a chronic inflammatory disease known to be mediated by numerous factors, among which endothelial dysfunction plays a critical role. Oscillatory shear stress induces endothelial cells to lose their anti-atherosclerotic properties and downregulates the expression of the innate protective transcription factor, Krüppel-like factor 2 (KLF2), which is typically upregulated in vascular endothelial cells in response to harmful stimuli. Oxidative stress and inflammation impair endothelial function and damage their survival. Oscillatory shear stress also promotes generation of reactive oxygen species and production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), thereby further promoting endothelial dysfunction and formation of atherosclerotic plaque. A major event in the development of atherosclerotic plaque is rolling and adhesion of monocytes to endothelial cells, which is mediated by adhesion molecules including vascular cellular adhesion molecule 1 and endothelial-selectin. Expression of these molecules is also upregulated by oscillatory shear stress. Estrogen has long been recognized as a protective agent against atherosclerosis, but the mechanisms through which estrogen receptors prevent atherogenesis remain unclear. In the present study, we investigated the role of the G-coupled protein estrogen receptor (GPR30) in oscillatory shear stress- induced endothelial dysfunction. We show that agonism of GPR30 by its specific agonist G1 prevented oscillatory shear stress -induced oxidative stress markers and production of inflammatory cytokines and adhesion molecules. As a result, GPR30 activation suppresses monocytes adhesion to endothelial cells. Furthermore, we demonstrate that GPR30 prevents oscillatory shear stress- induced downregulation of KLF2 via ERK5 pathway. These findings suggest that endothelial GPR30 is potential target to suppress oscillatory shear stress mediated atherogenesis.

The protective effects of azilsartan against oscillatory shear stress-induced endothelial dysfunction and inflammation are mediated by KLF6.

Atherosclerosis is a common cardiovascular disease with high morbidity and mortality. It is reported to be related to oscillatory shear stress (OSS)-induced endothelial dysfunction and excessive production of inflammatory factors. Azilsartan, a specific antagonist of the angiotensin II receptor, has been approved for the management of hypertensive subjects with diabetes mellitus type II (DMII). The present study will investigate the effects of azilsartan against OSS-induced endothelial dysfunction and inflammation, as well as the underlying mechanism. Cell viability was detected using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay were used to determine the expression levels of IL-6, TNF-α, IL-1β, VCAM-1, and ICAM-1 in human aortic endothelial cells (HAECs). Generation of reactive oxygen species (ROS) was measured using 2'-7'dichlorofluorescin diacetate (DCFH-DA) staining, and the level of reduced glutathione (GSH) was evaluated using a commercial kit. The adhesion of THP-1 monocytes to HAECs was evaluated using calcein-AM staining. The expression level of KLF6 was determined using qRT-PCR and Western blot analysis. According to the result of the MTT assay, 5 and 10 μM azilsartan were considered as the optimized concentrations applied in the present study. The elevated production of IL-6, TNF-α, and IL-1β, increased levels of ROS, decreased levels of reduced GSH, upregulated VCAM-1, ICAM-1, and E-selectin, and the aggravated adhesion of THP-1 cells to HAECs induced by OSS were all reversed by the introduction of azilsartan. The downregulation of KLF6 induced by OSS was significantly reversed by azilsartan. By knocking down the expression of KLF6, the suppressed adhesion of THP-1 cells to the HAECs, and the downregulation of VCAM-1 and ICAM-1 induced by azilsartan in OSS-stimulated HAECs were greatly reversed. The protective effects of azilsartan against OSS-induced endothelial dysfunction and inflammation might be mediated by KLF6.

Elevated ZIPK is required for TNF-α-induced cell adhesion molecule expression and leucocyte adhesion in endothelial cells.

Leucocyte adhesion to the vascular endothelium is a critical event in the early inflammatory response to infection and injury. This process is primarily regulated by the expression of cell adhesion molecules (CAMs) in endothelial cells. It has been well documented that tumor necrosis factor alpha (TNF-α) is a key regulator of CAM expression within this process, but its regulatory mechanism remains controversial. To investigate the scenario within this process, we assessed the role of zipper-interacting protein kinase (ZIPK), a serine/threonine kinase with multiple substrates, in CAM expression. We used TNF-α as inflammatory stimulator and found that ZIPK was integrated into the signaling regulation of TNF-α-mediated CAM expression. In human umbilical vein endothelial cells (HUVECs), TNF-α exposure led to significantly increased expression of both intercellular CAM-1 (ICAM-1) and vascular CAM-1 (VCAM-1), along with an increase in the adhesion of THP-1 monocytes to HUVECs. Simultaneously, ZIPK gene was also up-regulated at the transcription level. These effects were clearly inhibited by the ZIPK-specific inhibitor Tc-DAPK6 or small interfering RNA (siRNA) capable of specifically inhibiting ZIPK expression. We thus suggest that both ZIPK activation and ZIPK gene expression are necessary for TNF-α-mediated CAM expression and leucocyte adhesion. Interestingly, ZIPK inhibition also significantly suppressed TNF-α-induced nuclear factor kappa B (NF-κB) activation, indicating that TNF-α-mediated ZIPK expression functions upstream of NF-κB and CAM expression. We thus propose a TNF-α/ZIPK/NF-κB signaling axis for CAM expression that is necessary for leucocyte adhesion to endothelial cells. Our data in this study revealed a potential molecular target for exploring anti-inflammation drugs.

Impact of cigarette versus electronic cigarette aerosol conditioned media on aortic endothelial cells in a microfluidic cardiovascular model

Atherosclerosis is a complex process involving progressive pathological events, including monocyte adhesion to the luminal endothelial surface. We have developed a functional in vitro adhesion assay using BioFlux microfluidic technology to investigate THP-1 (human acute monocytic leukaemia cell) monocyte adhesion to human aortic endothelial cells (HAECs). The effect of whole smoke conditioned media (WSCM) generated from University of Kentucky reference cigarette 3R4F, electronic cigarette vapour conditioned media (eVCM) from an electronic nicotine delivery system (ENDS) product (Vype ePen) and nicotine on monocyte adhesion to HAECs was evaluated. Endothelial monolayers were grown in microfluidic channels and exposed to 0–1500 ng/mL nicotine or nicotine equivalence of WSCM or eVCM for 24 h. Activated THP-1 cells were perfused through the channels and a perfusion, adhesion period and wash cycle performed four times with increasing adhesion period lengths (10, 20, 30 and 40 min). THP-1 cell adhesion was quantified by counting adherent cells. WSCM induced dose-dependent increases in monocyte adhesion compared to vehicle control. No such increases were observed for eVCM or nicotine. Adhesion regulation was linked to increased ICAM-1 protein expression. Staining of ICAM-1 in HAECs and CD11b (MAC-1) in THP-1 cells demonstrated adhesion molecule co-localisation in BioFlux plates. The ICAM-1 adhesion response to WSCM was downregulated by transfecting HAECs with ICAM-1 siRNA. We conclude that the BioFlux system is able to model human monocyte adhesion to primary human endothelial cells in vitro and WSCM drives the greatest increase in monocyte adhesion via a mechanism involving endothelial ICAM-1 expression.

Detection of Protein Aggregation and Proteotoxicity Induced by Angiotensin II in Vascular Smooth Muscle Cells.

Disruption of protein quality control occurs with aging and cardiovascular pathologies including arterial stiffness and hypertension. Angiotensin II (Ang II) is believed to induce endoplasmic reticulum stress in vascular smooth muscle cells (VSMCs), thus contributing to vascular remodeling and dysfunction. However, whether Ang II increases formation of protein aggregates and mediates proteotoxicity in VSMCs remain obscure. Accordingly, this study aimed to establish a quantitative method of protein aggregate detection induced by Ang II and to investigate their potential involvement in inflammatory and senescence responses. Proteostat staining showed increased aggregate numbers per cell on Ang II exposure. Immunoblot analysis further showed an increase in preamyloid oligomer presence in a detergent insoluble protein fraction purified from VSMCs stimulated with Ang II. Moreover, these responses were attenuated by treatment with chemical chaperone, 4-phenylbutyrate. 4-phenylbutyrate further blocked Ang II-induced senescence associated β-galactosidase activity and THP-1 monocyte adhesion in VSMCs. These data suggest that Ang II induces proteotoxicity in VSMCs which likely contributes to aging and inflammation in the vasculature.

SARS-CoV-2 Spike Protein S1-Mediated Endothelial Injury and Pro-Inflammatory State Is Amplified by Dihydrotestosterone and Prevented by Mineralocorticoid Antagonism

Background: Men are disproportionately affected by the coronavirus disease-2019 (COVID-19) and experience higher mortality as compared to women. The vascular effects of androgen signaling and inflammatory cytokines in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mediated endothelial damage are not defined.Methods: Here, we determined the effects of SARS-CoV-2 spike protein-mediated endothelial damage under conditions of exposure to androgens and TNF-α. We tested the therapeutic effects of approved drugs: angiotensin receptor blockade by valsartan and mineralocorticoid receptor antagonism by spironolactone. Circulating endothelial injury markers VCAM-1 and E-selectin were measured in the plasma from men and women diagnosed with COVID-19.Findings: Exposure of endothelial cells (ECs) in vitro to androgen dihydrotestosterone (DHT) exacerbated spike protein S1-mediated endothelial injury transcripts for cell adhesion molecules E-selectin, VCAM-1, and ICAM-1 and anti-fibrinolytic PAI-1 (PInterpretation: Androgen exposure promotes spike protein-mediated endothelial injury by increasing markers of inflammation and thrombosis. A beneficial therapeutic effect of the FDA-approved drug spironolactone was observed, supporting a rationale for further evaluation as an adjunct treatment in COVID-19. Funding: This work was supported by the Michigan Biological Research Initiative on Sex Differences in Cardiovascular Disease, COVID-19 Ignitor Award, and University of Michigan Taubman Medical Research Institute.Declaration of Interests: No conflicts of interest, financial or otherwise, are declared by the author(s).Ethics Approval Statement: Study involving measurement of circulating endothelial injury markers in the plasma of COVID-19 patients was approved by the Institutional Review Board (HUM00180521) at the University of Michigan.

Mechanisms of endothelial cell activation by endocannabinoid 2-arachidonoylglycerol

Abstract Background Endothelial dysfunction promotes atherogenesis, vascular inflammation, and thrombus formation. Reendothelialization after angioplasty is required in order to restore vascular function and to prevent stent thrombosis. The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) is a known modulator of inflammation. Earlier studies have demonstrated the relevance of this endocannabinoid in human pathophysiology during coronary artery disease and in murine experimental atherogenesis. However, evidence on the impact of 2-AG on endothelial cell function remains scarce. Methods Endothelial repair was studied in two treatment groups of wildtype mice following electrical denudation of the common carotid artery. One group received the monoacylglycerol lipase (MAGL)-inhibitor JZL184, which impairs 2-AG degradation and thus causes elevated 2-AG levels, the other group received DMSO. The residual endothelial gap at five days was visualized by Evan's blue staining in either group. In vitro, the effect of 2-AG on human coronary artery endothelial cell (HCAEC) viability was assessed by an XTT-based assay. Endothelial activation was studied by an adhesion assay of THP-1 monocytes to 2-AG-preconditioned HCAEC. Activation of HCAEC adhesion molecules was characterized by flow cytometry. Results Elevated 2-AG levels significantly impaired reendothelialization in wildtype mice following electrical injury of the common carotid artery, resulting in a residual denudation at 5 days of 2291±286 μm vs. 1505±223 μm (n=18–19; p<0.05). In vitro, 2-AG significantly reduced viability of HCAEC at 24 hours (0.31±0.10 vs. 1.00±0.08; n=3; p<0.01). Finally, 2-AG promoted HCAEC activation resulting in a significant increase in THP-1 monocyte adhesion to HCAEC following pre-treatment of HCAEC with 2-AG (0.17±0.03 THP-1 cells per HCAEC vs. 0.07±0.01 THP-1 cells per HCAEC; n=3; p<0.05). Adhesion molecules E-selectin, ICAM-1 and VCAM-1, that are known to be regulated by 2-AG in the venous endothelium, remained unchanged in arterial endothelial cells. Besides, HCAEC migration, ROS-production, expression of NADPH oxidases and secretion of inflammatory cytokines were unaffected by 2-AG. Conclusion Elevated 2-AG levels hamper endothelial repair and impair HCAEC proliferation while facilitating adhesion of monocytes. Intriguingly, the underlying mechanisms in the arterial vascular bed appear distinct from venous endothelium. Given that 2-AG is elevated during coronary artery disease in humans, 2-AG might impair reendothelialization after angioplasty and thus impact on clinical outcomes. Funding Acknowledgement Type of funding source: None

Lymph Node Subcapsular Sinus Microenvironment-On-A-ChipModeling Shear Flow Relevant to Lymphatic Metastasis and Immune Cell Homing.

SummaryA lymph node sinus-on-a-chip adhesion microfluidic platform that recapitulates the hydrodynamic microenvironment of the lymph node subcapsular sinus was engineered. This device was used to interrogate the effects of lymph node remodeling on cellular adhesion in fluid flow relevant to lymphatic metastasis. Wall shear stress levels analytically estimated and modeled after quiescent and diseased/inflamed lymph nodes were experimentally recapitulated using a flow-based microfluidic perfusion system to assess the effects of physiological flow fields on human metastatic cancer cell adhesion. Results suggest that both altered fluid flow profiles and presentation of adhesive ligands, which are predicted to manifest within the lymph node subcapsular sinus as a result of inflammation-induced remodeling, and the presence of lymph-borne monocytic cells may synergistically contribute to the dynamic extent of cell adhesion in flow relevant to lymph node invasion by cancer and monocytic immune cells during lymphatic metastasis.

Gamma-Linolenic and Pinolenic Acids Exert Anti-Inflammatory Effects in Cultured Human Endothelial Cells Through Their Elongation Products.

Omega-3 fatty acids (FAs) from oily fish reduce cardiovascular disease. This may be partly due to modulation of endothelial cell (EC) inflammation. Fish stocks are declining and there is a need for sustainable alternative FAs. Gamma-linolenic acid (GLA) and pinolenic acid (PLA) are plant-derived FAs, which can fulfil this role. EA.hy926 cells are exposed GLA and PLA prior to stimulation with tumor necrosis factor (TNF)-α. GLA and PLA are incorporated into ECs, resulting in increases in long-chain derivatives produced by elongase 5, dihomo-gamma-linolenic acid (DGLA), and eicosatrienoic acid (ETA). Both GLA and PLA (50µm) decrease production of soluble intercellular adhesion molecule-1 (sICAM-1), monocyte chemoattractant protein 1 (MCP-1), and regulated on activation, normal T cell expressed and secreted (RANTES). However, decreases in these mediators are not seen after pre-treatment with GLA or PLA in elongase 5 silenced EA.hy926 cells. DGLA and ETA (10µm) decrease EC production of sICAM-1, MCP-1, RANTES, and IL-6. All FAs reduce adhesion of THP-1 monocytes to EA.hy926 cells. Both PLA (50µm) and ETA (10µm) decrease NFκBp65 phosphorylation. These effects suggest potential for GLA, PLA and their long-chain derivatives, DGLA and ETA, as sustainable anti-inflammatory alternatives to fish-derived FAs.

The endocannabinoid 2-arachidonoylglycerol inhibits endothelial function and repair

BackgroundEndothelial dysfunction promotes atherogenesis, vascular inflammation, and thrombus formation. Reendothelialization after angioplasty is required in order to prevent stent failure. Previous studies have highlighted the role of 2-arachidonoylglycerol (2-AG) in murine experimental atherogenesis and in human coronary artery disease. However, the impact of 2-AG on endothelial repair and leukocyte–endothelial cell adhesion is still unknown. MethodsEndothelial repair was studied in two treatment groups of wildtype mice following electrical injury of the common carotid artery. One group received the monoacylglycerol lipase (MAGL)-inhibitor JZL184, which impairs 2-AG degradation and thus causes elevated 2-AG levels, the other group received DMSO (vehicle). The effect of 2-AG on human coronary artery endothelial cell (HCAEC) viability, leukocyte–endothelial cell adhesion, surface expression of adhesion molecules, and expression of endothelial NO synthase (NOS3) was studied in vitro. ResultsElevated 2-AG levels significantly impaired reendothelialization in wildtype mice following electrical injury of the common carotid artery. In vitro, 2-AG significantly reduced viability of HCAEC. Additionally, 2-AG promoted adhesion of THP-1 monocytes to HCAEC following pre-treatment of the HCAEC with 2-AG. Adhesion molecules (E-selectin, ICAM-1 and VCAM-1) remained unchanged in arterial endothelial cells, whereas 2-AG suppressed the expression of NOS3 in HCAEC. Conclusion and translational aspectElevated 2-AG levels hamper endothelial repair and HCAEC proliferation, while simultaneously facilitating leukocyte–endothelial cell adhesion. Given that 2-AG is elevated in patients with coronary artery disease and non-ST-segment elevation myocardial infarction, 2-AG might decrease reendothelialization after angioplasty and thus impact the clinical outcomes.

Covid-19: Acts of omission

<h3>Background</h3> Baricitinib is a Janus kinase (JAK)1/JAK2 inhibitor approved for the treatment of rheumatoid arthritis (RA)^1-2. STAT proteins bind JAK kinase dimers coupled with cytokine receptors and regulate gene transcription. The JAK/STAT system is responsible for the intracellular signaling of different cytokines contributing to the activation process of the monocyte lineage, therefore the use of JAK inhibitors can affect cell functionality^3-6. <h3>Objectives</h3> The aim of the present study was to verify the effects of baricitinib on STAT phosphorylation in peripheral mononuclear cells (PBMCs) of RA patients and to evaluate any correlation between STAT phosphorylation and response to therapy. <h3>Methods</h3> At baseline (BL) and after 4 weeks (w4) of treatment, we evaluated patients’ disease activity (DAS28_PCR, CDAI and SDAI), dividing them into responders and non-responders according to the Minimal Clinically Important Difference for DAS28_PCR (1.2 points) at w4. The phosphorylation of STAT1, STAT4 and STAT5 was analyzed at BL and w4 in gated monocytes, Treg, CD8 + and CD4 + lymphocytes from 4 responder and 4 non-responder patients through flow cytometry, at basal conditions and after IL2, IFNα and IL6 stimulation. <h3>Results</h3> Baseline clinical and demographic characteristics of patients are reported in Table 1. We showed that monocyte count decreased from BL to w4 mostly in responders. Basal pSTAT1 phosphorylation tent to be higher in monocytes of non-responder patients; after 4 weeks of treatment, the reduction of the cytokine-induced pSTAT1 was significantly greater in monocytes from responders compared to non-responders (Figure 1). The phosphorylation of STAT4 and STAT5 was not affected by treatment in any cell type and at any time point. We further studied the STAT1 phosphorylation pathway isolating the effect of stimulation with IFNα and stratifying monocytes according to their surface marker expression of CD14 and CD16 in classical, intermediate and non-classical. We observed the same pattern with a significant greater reduction of pSTAT1 in monocytes from responder patients, compared to non-responders, after the treatment with baricitinib. <h3>Conclusion</h3> These results may suggest that monocyte count and STAT1 phosphorylation in circulating monocytes could represent early markers of response to baricitinib therapy. <h3>References</h3> [1]Gadina M, et al. Janus kinases to jakinibs: from basic insights to clinical practice. Rheumatology (Oxford). 2019 [2]Gadina M, et al. Translating JAKs to Jakinibs. J Immunol. 2020 [3]Kubo S, et al. The JAK inhibitor, tofacitinib, reduces the T cell stimulatory capacity of human monocyte-derived dendritic cells. Ann Rheum Dis. 2014 [4]Kubo S, et al. Janus Kinase Inhibitor Baricitinib Modulates Human Innate and Adaptive Immune System. Front Immunol. 2018 [5]Ikari Y, et al. Peficitinib Inhibits the Chemotactic Activity of Monocytes via Proinflammatory Cytokine Production in Rheumatoid Arthritis Fibroblast-Like Synoviocytes. Cells. 2019 [6]Yang X, et al. Tofacitinib inhibits ox-LDL-induced adhesion of THP-1 monocytes to endothelial cells. Artif Cells Nanomed Biotechnol. 2019 <h3>Disclosure of Interests</h3> Cristina Garufi Consultant of: Lilly, Gloria Tucci: None declared, Ilenia Pacella: None declared, Marta Zagaglioni: None declared, Alessandra Pinzon Grimaldos: None declared, Fulvia Ceccarelli: None declared, Silvia Piconese: None declared, Francesca Romana Spinelli Consultant of: Lilly, Fabrizio Conti Consultant of: Lilly

Role of growth hormone signaling pathways in the development of atherosclerosis

ObjectiveThe direct actions of growth hormone (GH) in the development of atherosclerosis are unclear. The goal of this study was to characterize GH-induced changes in expression of signaling pathway elements and other proteins that may be related to atherosclerosis. MethodsHuman umbilical vein endothelial cells (HUVEC) and THP-1, a human acute monocytic leukemia cell line, were stimulated by exposure to 10−9 M or 10−8 M human GH with or without pretreatment with a mitogen-activated protein kinase kinase (MEK) 1 inhibitor. Levels of transcripts encoding vascular cell adhesion molecule (VCAM) -1, E-selectin, monocyte chemotactic protein (MCP-1), interleukin (IL) -6, and IL-8 were investigated by reverse transcription (RT) -PCR. For the quantitative adhesion assay, THP-1 cells or human primary monocytes were fluorescently labeled with 3’-O-acetyl-2′,7′-bis(carboxyethyl) -4 diacetoxymethyl ester (BCECF/AM). HUVEC treated with human GH were co-incubated with BCECF-labeled THP-1 cells. One hour later, the number of BCECF-labeled THP-1 cells was assessed. An equivalent experiment was performed using BCECF-labeled primary monocytes, and the number of monocytes adhering to HUVEC was counted. ResultsTreatment with hGH increased the levels of E-selectin- and VCAM-1-encoding mRNAs in HUVEC. This effect was attenuated by pretreatment with a MEK1 inhibitor. Furthermore, hGH treatment increased adhesion of BCECF-labeled THP-1 cells or primary monocytes to HUVEC, and this effect was attenuated by pretreatment with a MEK1 inhibitor. ConclusionsVCAM-1 and E-selectin expression was stimulated by GH via the mitogen-activated protein kinase pathway, resulting in augmented adhesion of THP-1 cells and monocytes to HUVEC. These data suggested that GH directly stimulates the development of atherosclerosis.

β-Endorphin Mediates the Development and Instability of Atherosclerotic Plaques.

β-Endorphin, an endogenous opioid peptide, and its μ-opioid receptor are expressed in brain, liver, and peripheral tissues. β-Endorphin induces endothelial dysfunction and is related to insulin resistance. We clarified the effects of β-endorphin on atherosclerosis. We assessed the effects of β-endorphin on the inflammatory response and monocyte adhesion in human umbilical vein endothelial cells (HUVECs), foam cell formation, and the inflammatory phenotype in THP-1 monocyte-derived macrophages, and migration and proliferation of human aortic smooth muscle cells (HASMCs) in vitro. We also assessed the effects of β-endorphin on aortic lesions in Apoe−/− mice in vivo. The μ-opioid receptor (OPRM1) was expressed in THP-1 monocytes, macrophages, HASMCs, HUVECs, and human aortic endothelial cells. β-Endorphin significantly increased THP-1 monocyte adhesion to HUVECs and induced upregulation of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin via nuclear factor-κB (NF-κB) and p38 phosphorylation in HUVECs. β-Endorphin significantly increased HUVEC proliferation and enhanced oxidized low-density lipoprotein-induced foam cell formation in macrophages. β-Endorphin also significantly shifted the macrophage phenotype to proinflammatory M1 rather than anti-inflammatory M2 via NF-κB phosphorylation during monocyte-macrophage differentiation and increased migration and apoptosis in association with c-jun-N-terminal kinase, p38, and NF-κB phosphorylation in HASMCs. Chronic β-endorphin infusion into Apoe−/− mice significantly aggravated the development of aortic atherosclerotic lesions, with an increase in vascular inflammation and the intraplaque macrophage/smooth muscle cell ratio, an index of plaque instability. Our study provides the first evidence that β-endorphin contributes to the acceleration of the progression and instability of atheromatous plaques. Thus, μ-opioid receptor antagonists may be useful for the prevention and treatment of atherosclerosis.

Maslinic acid suppresses macrophage foam cells formation: Regulation of monocyte recruitment and macrophage lipids homeostasis

The transformation of macrophages to foam cells is a critical component in atherosclerotic lesion formation. Maslinic acid (MA), a novel natural pentacyclic triterpene, has cardioprotective and anti-inflammatory properties. It is hypothesized that MA can suppress monocyte recruitment to endothelial cells and inhibit macrophage foam cells formation. Previous study shows that MA inhibits inflammatory effects induced by sPLA2-IIA, including foam cells formation. This study elucidates the regulatory effect of MA in monocyte recruitment, macrophage lipid accumulation and cholesterol efflux. Our findings demonstrate that MA inhibits THP-1 monocyte adhesion to HUVEC cells in a TNFα-dependent and independent manner, but it induces trans-endothelial migration marginally at low concentration. MA down-regulates both gene and protein expression on VCAM-1 and MCP-1 in HUVECs. We further showed that MA suppresses macrophage foam cells formation, as indicated from the Oil-Red-O staining and flow cytometric analysis of intracellular lipids accumulation. The effects observed may be attributed to the antioxidant properties of MA where it was shown to suppress CuSO4-induced lipid peroxidation. MA inhibits scavenger receptors SR-A and CD36 expression while enhancing cholesterol efflux. MA enhances cholesterol efflux transporters ABCA1 and ABCG1 genes expression marginally without inducing its protein expression. In this study, MA was shown to target important steps that contribute to foam cell formation, including its ability in reducing monocytes adhesion to endothelial cells and LDL peroxidation, down-regulating scavenger receptors expression as well as enhancing cholesterol efflux, which might be of great importance in the context of atherosclerosis prevention and treatment.

RETRACTED: Levetiracetam inhibits THP-1 monocyte chemotaxis and adhesion via the synaptic vesicle 2A

Long-term therapy with older antiepileptic drugs (AEDs), but not levetiracetam (LEV), may increase the risk of atherosclerosis (AS), suggesting that LEV may have a potential anti-AS effect. The synaptic vesicle 2A (SV2A) is known to the specific binding site of LEV. Numerous studies have documented that SV2A is a membrane protein specifically expressed in nervous system. Interestingly, our previous research showed that SV2A also existed in human CD8+ T lymphocytes. Therefore, we hypothesized that LEV was associated with decreased risk of AS by regulating monocytes chemotaxis and adhesion. We showed that SV2A protein were detected in THP-1 human monocytic leukemia cells. LEV (300μM) inhibited the chemotaxis and adhesion of THP-1cells after transfection with plasmids expressing SV2AWT, but not SV2AR383Q which was a known functional mutation site of human SV2A. Furthermore, RT-PCR and western blot analysis demonstrated that LEV (300μM) decreased the expression level of chemokine-related receptors (CX3CL1, CCR1, CCR2, and CCR5),and reduced levels of phosphorylated AKT (p-AKT) in THP-1cells with SV2AWT expressing plasmids. Taken together, these findings indicated that LEV has an inhibitory effect on THP-1 monocyte adhesion and chemotaxis, suggesting that SV2A may serve as a novel therapeutic target to prevent AS.

Astragaloside IV suppresses inflammatory response via suppression of NF-κB, and MAPK signalling in human bronchial epithelial cells

Context Astragaloside IV isolated from Astragalus membranaceus (Fisch.), which was reported to have anti-tumor, anti-asthma, and suppressed cigarette smoke-induced lung inflammation in mice. Objectives This study investigated whether astragaloside IV reduced the expression of inflammatory mediators and oxidative stress in BEAS-2B cells. Methods BEAS-2B cells treated with astragaloside IV, and then stimulated with TNF-α or TNF-α/IL-4. The levels of cytokine and chemokine were analysed with ELISA and real-time PCR. Results Astragaloside IV significantly inhibited the levels of CCL5, MCP-1, IL-6 and IL-8. Astragaloside IV also reduced ICAM-1 expression for blocked THP-1 monocyte adhesion to BEAS-2B cells. Furthermore, astragaloside IV attenuated the phosphorylation of MAPK, and reduced the translocation of p65 into the nucleus. Astragaloside IV could increase the expression of HO-1 and Nrf2 for promoting the oxidant protective effect. Conclusion Aastragaloside IV has an anti-inflammatory and oxidative effect via regulated NF-κB, MAPK and HO-1/Nrf2 signalling pathways in human bronchial epithelial cells.