Increased Monocyte Adhesion Research Articles

The microbial metabolite p-cresol compromises the vascular barrier and induces endothelial cytotoxicity and inflammation in a 3D human vessel-on-a-chip

Increased protein-bound uremic toxins (PBUTs) in patients with chronic kidney disease (CKD) are associated with cardiovascular diseases (CVDs); however, whether retention of PBUTs causes CVD remains unclear. Previous studies assessing the impacts of PBUTs on the vasculature have relied on 2D cell cultures lacking in vivo microenvironments. Here, we investigated the impact of various PBUTs (p-cresol (PC), indoxyl sulfate (IS), and p-cresyl sulfate (PCS)) on microvascular function using an organ-on-a-chip (OOC). Human umbilical vein endothelial cells were used to develop 3D vessels. Chronic exposure to PC resulted in significant vascular leakage compared with controls, whereas IS or PCS treatment did not alter the permeability of 3D vessels. Increased permeability induced by PC was correlated with derangement of cell adherens junction complex, vascular endothelial (VE)-cadherin and filamentous (F)-actin. Additionally, PC decreased endothelial viability in a concentration-dependent manner with a lower IC50 in 3D vessels than in 2D cultures. IS slightly decreased cell viability, while PCS did not affect viability. PC induced inflammatory responses by increasing monocyte adhesion to endothelial surfaces of 3D vessels and IL-6 production. In conclusion, this study leveraged an OOC to determine the diverse effects of PBUTs, demonstrating that PC accumulation is detrimental to ECs during kidney insufficiency.

MMP-9로 자른 피브로넥틴에 의한 뇌종양세포 U87MG Cells와 단핵구 U937 Cells간의 부착증가 효과

This study investigated the pivotal role of fibronectin cleaved by MMP-9 in enhancing the attachment of monocytes to tumor cells. To determine whether MMP-9 induces changes in the extracellular matrix fibronectin of tumor cells and increases monocyte adhesion, human glioblastoma U87MG cells expressing MMP-9 were established by transfection. The human monocyte U937 cells adhered significantly to U87MG cells expressing MMP-9, however, they did not adhere to U87MG cells that did not express MMP-9. To directly demonstrate the role of MMP-9-cleaved fibronectin in increasing monocyte U937 cell adhesion, the attachment of monocyte U937 cells to fibronectin treated with MMP-9 was confirmed. These findings suggest that MMP-9 alters the extracellular matrix, specifically fibronectin, and plays a crucial role in mediating the adhesion between immune and tumor cells.

Lipid Variability Induces Endothelial Dysfunction by Increasing Inflammation and Oxidative Stress.

This study investigates the impact of fluctuating lipid levels on endothelial dysfunction. Human aortic and umbilical vein endothelial cells were cultured under varying palmitic acid (PA) concentrations: 0, 50, and 100 μM, and in a variability group alternating between 0 and 100 μM PA every 8 hours for 48 hours. In the lipid variability group, cells were exposed to 100 μM PA during the final 8 hours before analysis. We assessed inflammation using real-time polymerase chain reaction, Western blot, and cytokine enzyme-linked immunosorbent assay (ELISA); reactive oxygen species (ROS) levels with dichlorofluorescin diacetate assay; mitochondrial function through oxygen consumption rates via XF24 flux analyzer; and endothelial cell functionality via wound healing and cell adhesion assays. Cell viability was evaluated using the MTT assay. Variable PA levels significantly upregulated inflammatory genes and adhesion molecules (Il6, Mcp1, Icam, Vcam, E-selectin, iNos) at both transcriptomic and protein levels in human endothelial cells. Oscillating lipid levels reduced basal respiration, adenosine triphosphate synthesis, and maximal respiration, indicating mitochondrial dysfunction. This lipid variability also elevated ROS levels, contributing to a chronic inflammatory state. Functionally, these changes impaired cell migration and increased monocyte adhesion, and induced endothelial apoptosis, evidenced by reduced cell viability, increased BAX, and decreased BCL2 expression. Lipid variability induce endothelial dysfunction by elevating inflammation and oxidative stress, providing mechanistic insights into how lipid variability increases cardiovascular risk.

In vitro and in vivo exposure of endothelial cells to dibutyl phthalate promotes monocyte adhesion

The effect of endothelial cells’ exposure to dibutyl phthalate (DBP) on monocyte adhesion is largely unknown. We evaluated monocyte adhesion to DBP-exposed endothelial cells by combining three approaches: short-term exposure (24 h) of EA.hy926 cells to 10−6, 10−5, and 10−4 M DBP, long-term exposure (12 weeks) of EA.hy926 cells to 10−9, 10−8, and 10−7 M DBP, and exposure of rats (28 and 90 days) to 100, 500, and 5000 mg DBP/kg food. Monocyte adhesion to human EA.hy926 and rat aortic endothelial cells, expression of selected cellular adhesion molecules and chemokines, and the involvement of extracellular signal-regulated kinase 1/2 (ERK1/2) were analyzed. We observed increased monocyte adhesion to DBP-exposed EA.hy926 cells in vitro and to rat aortic endothelium ex vivo. ERK1/2 inhibitor prevented monocyte adhesion to DBP-exposed EA.hy926 cells in short-term exposure experiments. Increased ERK1/2 phosphorylation in rat aortic endothelium and transient decrease in ERK1/2 activation following long-term exposure of EA.hy926 cells to DBP were also observed. In summary, exposure of endothelial cells to DBP promotes monocyte adhesion, thus suggesting a possible role for this phthalate in the development of atherosclerosis. ERK1/2 signaling could be the mediator of monocyte adhesion to DBP-exposed endothelial cells, but only after short-term high-level exposure.

Modeling Foam Cell Formation in A Hydrogel-Based 3D-Intimal Model: A Study of The Role of Multi-Diseases During Early Atherosclerosis.

Monocyte recruitment and transmigration are crucial in atherosclerotic plaque development. The multi-disease complexities aggravate the situation and continue to be a constant concern for understanding atherosclerosis plaque development. Herein, a 3D hydrogel-based model that integrates disease-induced microenvironments is sought to be designed, allowing us to explore the early stages of atherosclerosis, specifically examining monocyte fate in multi-disease complexities. As a proof-of-concept study, murine cells are employed to develop the model. The model is constructed with collagen embedded with murine aortic smooth muscle cells and a murine endothelial monolayer lining. The model achieves in vitro disease complexities using external stimuli such as glucose and lipopolysaccharide (LPS). Hyperglycemia exhibits a significant increase in monocyte adhesion but no enhancement in monocyte transmigration and foam cell conversion compared to euglycemia. Chronic infection achieved by LPS stimulation results in a remarkable augment in initial monocyte attachment and a significant increment in monocyte transmigration and foam cells in all concentrations. Moreover, the model exhibits synergistic sensitivity under multi-disease conditions such as hyperglycemia and infection, enhancing initial monocyte attachment, cell transmigration, and foam cell formation. Additionally, western blot data prove the enhanced levels of inflammatory biomarkers, indicating the model's capability to mimic disease-induced complexities during early atherosclerosis progression.

P2Y2-P2X7 receptors cross-talk in primed mesenteric endothelial cells upregulates NF-κB signaling favoring mononuclear cell adhesion in schistosomiasis.

Schistosomiasis is an intravascular infectious disease that impacts over 200 million people globally. In its chronic stage, it leads to mesenteric inflammation with significant involvement of monocytes/macrophages. Endothelial cells lining the vessel lumens play a crucial role, and mount of evidence links this disease to a downregulation of endoprotective cell signaling favoring a primed and proinflammatory endothelial cell phenotype and therefore the loss of immunovascular homeostasis. One hallmark of infectious and inflammatory conditions is the release of nucleotides into the extracellular milieu, which, in turn, act as innate messengers, activating purinergic receptors and triggering cell-to-cell communication. ATP influences the progression of various diseases through P2X and P2Y purinergic receptor subtypes. Among these receptors, P2Y2 (P2Y2R) and P2X7 (P2X7R) receptors stand out, known for their roles in inflammation. However, their specific role in schistosomiasis has remained largely unexplored. Therefore, we hypothesized that endothelial P2Y2R and P2X7R could contribute to monocyte adhesion to mesenteric endothelial cells in schistosomiasis. Using a preclinical murine model of schistosomiasis associated with endothelial dysfunction and age-matched control mice, we showed that endothelial P2Y2R and P2X7R activation increased monocyte adhesion to cultured primary endothelial cells in both groups. However, a distinct upregulation of endothelial P2Y2R-driven canonical Ca2+ signaling was observed in the infected group, amplifying adhesion. In the control group, the coactivation of endothelial P2Y2R and P2X7R did not alter the maximal monocyte adhesion induced by each receptor individually. However, in the infected group, this coactivation induced a distinct upregulation of P2Y2R-P2X7R-driven canonical signaling, IL-1β release, and VCAM-1 expression, with underlying mechanisms involving inflammasome and NF-κB signaling. Therefore, current data suggest that schistosomiasis alters endothelial cell P2Y2R/P2X7R signaling during inflammation. These discoveries advance our understanding of schistosomiasis. This intricate interplay, driven by PAMP-triggered endothelial P2Y2R/P2X7R cross-talk, emerges as a potential key player in the mesenteric inflammation during schistosomiasis.

A three-dimensional vessel-on-chip model to study Puumala orthohantavirus pathogenesis.

Puumala orthohantavirus (PUUV) infection in humans can result in hemorrhagic fever with renal syndrome. Endothelial cells (ECs) are primarily infected with increased vascular permeability as a central aspect of pathogenesis. Historically, most studies included ECs cultured under static two-dimensional (2D) conditions, thereby not recapitulating the physiological environment due to their lack of flow and inherent pro-inflammatory state. Here, we present a high-throughput model for culturing primary human umbilical vein ECs in 3D vessels-on-chip in which we compared host responses of these ECs to those of static 2D-cultured ECs on a transcriptional level. The phenotype of ECs in vessels-on-chip more closely resembled the in vivo situation due to higher similarity in expression of genes encoding described markers for disease severity and coagulopathy, including IDO1, LGALS3BP, IL6 and PLAT, and more diverse endothelial-leukocyte interactions in the context of PUUV infection. In these vessels-on-chip, PUUV infection did not directly increase vascular permeability, but increased monocyte adhesion. This platform can be used for studying pathogenesis and assessment of possible therapeutics for other endotheliotropic viruses even in high biocontainment facilities.

Epigenetically-driven persistence of inflammatory and oxidative phenotypes in adolescents born to women with gestational diabetes

Abstract Background According to the theory of developmental origins of health and disease1, environmental changes during the in-utero period can permanently affect health and vulnerability to diseases later in life. As a matter of fact, the offspring of women with gestational diabetes (GD) exhibit a higher risk for developing obesity, type 2 diabetes (T2D), and atherosclerotic cardiovascular disease (ASCVD) than those individuals with a family history of diabetes2–4. Importantly, epigenetics has emerged as a crucial determinant for this transmission across generations5–8. Mixed Lineage Leukemia 1 (MLL1) histone methyltransferase activates inflammatory and oxidative pathways through trimethylation of histone 3 at lysine 4 amino (H3K4me3)9. Purpose To investigate whether such epigenetic mark regulates genes triggering inflammation and oxidative stress in GD women, and its potential transmission and persistence in their offspring. Methods We isolated peripheral blood mononuclear cells (PBMCs), fetal cord blood mononuclear cells (CBMCs), and umbilical cord vein endothelial cells (HUVECs) from GD and control pregnant women, as well as PBMCs and plasma samples from a retrospective cohort of adolescents (12-16 years old) born to control or GD women. Gene and protein expressions were investigated by RT-PCR, immunoblotting and/or confocal microscopy. H3K4me3 levels on NF-kB p65 and NADPH oxidase isoform 4 (NOX4) promoter regions were assessed by chromatin immunoprecipitation (ChIP) and RT-PCR. Mechanistic studies were performed in control- and GD-HUVECs using the MLL1 inhibitor MM-102 to assess downstream inflammatory mediators and superoxide anion (O2-) generation by electron spin resonance (ESR) spectroscopy. Results Our data showed upregulation of MLL1 and H3K4me3 levels in PBMCs, HUVECs, and CBMCs isolated from GD as compared to control women. ChIP analysis revealed H3K4me3 enrichment in the promoter region of the inflammatory and oxidative master regulatory genes NF-kB p65 and NOX4, respectively, in these cells. We also found an upregulation of NF-kB p65 target genes IL-6, MCP-1, ICAM-1, and VCAM-1, and increased monocyte adhesion and O2- production. The inhibition of MLL1 was able to reduce H3K4me3 levels blunting the inflammatory and oxidative phenotypes in GD-HUVECs. Interestingly enough, we demonstrated a persistent increase of H3K4me3 in the NF-kB p65 promoter region of PBMCs together with an elevation of inflammatory markers in plasma from adolescents born to GD women. Conclusions Our results suggest that GD may induce inflammation and oxidative stress through MLL1-dependent H3K4me3 mechanisms. Such epigenetic mark is transmitted to the offspring and, most importantly, can still be found in adolescence as a pro-inflammatory trigger. These findings pave the way for pharmacological reprogramming of adverse histone modifications to counteract early abnormal phenotypes in GD offspring which may accelerate ASCVD burden.

ScRNA-seq and flow cytometry based analysis reveals that chronic heart failure associates with enhanced activation and clonal expansion of T cells with predictive autoreactive capacity

Abstract Background Chronic heart failure (HF) is marked by adverse remodelling and chronic inflammation causing impaired function of the heart and poor prognosis. While much is known about the immune response immediately after MI, little is known of the role of the immune system in chronic HF. Our aim is to elucidate the response of T cells to HF. Methods and Results In a pilot study assessing healthy controls (HC) and HF patients (n=16), scRNA-seq showed an enhanced T cell activation profile in HF patients combined with an increased capacity for adhesion and antigen-presentation on monocytes. A confirmation cohort of n=180 HC and HF patients using flow cytometry of peripheral blood confirmed an increase of T cell memory/effector cells (HF=65%, HC=53% activated T cells), representing activated and stimulated T cells, and a reduction in the non-active naïve T cell compartment in HF patients. Correspondingly, APCs showed a higher immune stimulatory profile (e.g. HLA-DR, ICAM-1, TREM-1). Immune secretome analysis further supports the chronic inflammatory phase by significantly increased serum levels of sICAM-1, IL6 and TNFRI. Given that T cells showed strong activation profiles in HF patients, we investigated whether T cells are preferentially clonally expanded in the same patients. Bulk TCR sequencing revealed that TCR diversity was significantly reduced (p=0.03) along with clonal expansion of T cells in HF patients. To assess what these clonally expanding T cells might be targeting, we utilized epitope prediction tools to identify 22 unique human-derived epitopes found only in HF patients. ScRNA-seq of CD45+ sorted cardiac-derived and circulating cells of the same HF patient (N=1) versus healthy controls (n=4) showed clonal expansion of TCRs in the cardiac tissue and blood in HF and epitope prediction confirmed epitopes found in bulk TCR sequencing. We then assessed the T cell response in a time course post-myocardial infarction in mice with scRNA-seq. We found an accumulation of specifically pro-inflammatory (Th17 T cells in the heart at days 14, 28 and 48 post-MI. Dimensional reduction analysis of T cells from all time points showed that T cells appearing at days 28 and 48 post-MI were more pro-inflammatory than at the earlier time points. To assess the impact of immune dysregulation in HF on cardiac cells, we cultured the secretome of HF or HC immune cells with endothelial cells. We found that HF secretomes decreased viability of endothelial cells and increased monocyte adhesion to stimulated endothelial cells (p=0.005). Additionally, the immune regulatory molecule PDL-1 was downregulated in HF secretome treated endothelial cells (p=0.014). Conclusion This study suggests that chronic HF may drive persistent T cell activation and clonal expansion, with potential cardiac-relevant autoimmune implications causing or promoting adverse remodelling. These data also provide additional insights for therapeutic cardio-immunological interventions.

PCSK9 activation promotes early atherosclerosis in a vascular microphysiological system.

Atherosclerosis is a primary precursor of cardiovascular disease (CVD), the leading cause of death worldwide. While proprotein convertase subtilisin/kexin 9 (PCSK9) contributes to CVD by degrading low-density lipoprotein receptors (LDLR) and altering lipid metabolism, PCSK9 also influences vascular inflammation, further promoting atherosclerosis. Here, we utilized a vascular microphysiological system to test the effect of PCSK9 activation or repression on the initiation of atherosclerosis and to screen the efficacy of a small molecule PCSK9 inhibitor. We have generated PCSK9 over-expressed (P+) or repressed (P-) human induced pluripotent stem cells (iPSCs) and further differentiated them to smooth muscle cells (viSMCs) or endothelial cells (viECs). Tissue-engineered blood vessels (TEBVs) made from P+ viSMCs and viECs resulted in increased monocyte adhesion compared to the wild type (WT) or P- equivalents when treated with enzyme-modified LDL (eLDL) and TNF-α. We also found significant viEC dysfunction, such as increased secretion of VCAM-1, TNF-α, and IL-6, in P+ viECs treated with eLDL and TNF-α. A small molecule compound, NYX-1492, that was originally designed to block PCSK9 binding with the LDLR was tested in TEBVs to determine its effect on lowering PCSK9-induced inflammation. The compound reduced monocyte adhesion in P+ TEBVs with evidence of lowering secretion of VCAM-1 and TNF-α. These results suggest that PCSK9 inhibition may decrease vascular inflammation in addition to lowering plasma LDL levels, enhancing its anti-atherosclerotic effects, particularly in patients with elevated chronic inflammation.

Abstract P3069: ScRNA-seq And Flow Cytometry Based Analysis Reveals That Chronic Heart Failure Associates With Enhanced Activation And Clonal Expansion Of T Cells With Predictive Autoreactive Capacity

Background: Heart failure (HF) is marked by adverse remodelling and chronic inflammation causing impaired function of the heart and poor prognosis. While much is known about the immune response immediately after MI, little is known of the role of the immune system in chronic HF. Our aim is to elucidate the response of T cells to HF. Methods and Results: In a pilot study assessing healthy controls (HC) and HF patients (n=16), scRNA-seq showed an enhanced T cell activation profile in HF patients. A confirmation cohort of n=180 HC and HF patients using flow cytometry of immune cells confirmed an increase of T cell memory/effector cells (HF=65%, HC=53% activated T cells). Correspondingly, circulating antigen presenting cells showed augmented inflammatory profiles (ICAM-1, TREM-1). Given that T cells showed strong activation profiles in HF patients, we investigated whether T cells are preferentially clonally expanded in the same patients. Bulk T cell receptor (TCR) sequencing revealed that TCR diversity was significantly reduced (p=0.03) along with clonal expansion of T cells in HF patients. To assess what T cells might be targeting, we utilized epitope prediction tools to identify 22 unique human-derived epitopes found only in HF patients. ScRNA-seq of CD45+ sorted cardiac-derived and circulating cells of the same HF patient (N=1) versus healthy controls (n=4) showed clonal expansion of TCRs in the cardiac tissue and blood in HF and epitope prediction confirmed epitopes found in bulk TCR sequencing. When modelling this in mice, a scRNA-seq post-MI time course also found an accumulation of specifically pro-inflammatory (Th17) T cells in the heart at days 14, 28 and 48 post-MI. To assess the impact of immune dysregulation in HF on cardiac cells, we cultured the secretome of HF or HC immune cells with endothelial cells and found decreased viability of endothelial cells and increased monocyte adhesion to stimulated endothelial cells (p=0.005). Conclusion: This study suggests that chronic HF may drive persistent T cell activation and clonal expansion, with potential cardiac-relevant autoimmune implications causing or promoting adverse remodelling. These data also provide additional insights for therapeutic cardio-immunological interventions.

#4451 MONOCYTE MIGRATION AND ACTIVATION IN ANCA-ASSOCIATED GLOMERULONEPHRITIS

Abstract Background and Aims Glomerular injury in Anti-Neutrophil Cytoplasmic Antibodies (ANCA)-associated glomerulonephritis (AGN) is associated with macrophage infiltration. To date, their role and origin remain to be fully elucidated. Activated blood monocytes likely transmigrate and differentiate into kidney macrophages depending on the local microenvironment. Even in remission, some studies indicate sustained monocyte activation and upregulation of adhesion markers. The aim of this study was to research the intrinsic migration capacity of monocytes in AGN. Targeting monocyte migration might open new therapeutic avenues in the treatment of AGN. Method Transendothelial migration of monocytes was tested in AGN patients with active disease (n = 2), in remission (n = 8) and healthy controls (n = 6). To assess their adhesive and migratory capacity, freshly isolated CD14 positive monocytes were added to confluent TNF-α or IL1β overnight-stimulated Human Aortic Endothelial Cell (HAEC) for 30 minutes and subsequently fixed. Monocyte adhesion and transmigration was visualized by phase-contrast microscopy and quantified using ImageJ. To unravel the potential mechanisms driving these differences in monocyte migration, we performed bulk RNA-sequencing of monocytes from AGN patients with active (n = 4) and stable (n = 10) disease, and healthy controls (HC) (n = 6). Results Monocyte adhesion, but not migration, was significantly increased during active disease, independently of the stimuli used to mimic the pro-inflammatory phenotype of endothelial cells (EC) (Figure 1). During remission, decreased adhesion was found on the IL1β-stimulated ECs. While CD11b mRNA expression was upregulated, CD11a expression was downregulated in monocytes from active AGN patients compared to HC. Two genes involved in paracellular monocyte transmigration (JAML, PECAM-1) were significantly decreased. Most AGN patients were treated with corticosteroids at time of experiments. Conclusion These results suggest a remarkable increase in monocyte adhesion, but lower intrinsic migration capacity, in a 30 minute time-frame during active AGN. Our findings on migration are surprising in the light of theorized enhanced monocyte extravasation towards diseased AGN kidneys. Therefore, it could be speculated that after a longer period of time, a subset of the adherent monocytes would ultimately migrate and differentiate into kidney macrophages. In remission, long-term immunosuppressive treatment or chronic inflammation might decrease monocyte adhesion. This was the first ex-vivo study to research monocyte migration in AGN, further research is required to validate findings and to develop new therapies targeting monocyte migration.

LncRNA NIPA1-SO confers atherosclerotic protection by suppressing the transmembrane protein NIPA1

Long non-coding RNAs (lncRNAs) are emerging as important players in gene regulation and cardiovascular diseases. However, the roles of lncRNAs in atherosclerosis are poorly understood. In the present study, we found that the levels of NIPA1-SO were decreased while those of NIPA1 were increased in human atherosclerotic plaques. Furthermore, NIPA1-SO negatively regulated NIPA1 expression in human umbilical vein endothelial cells (HUVECs). Mechanistically, NIPA1-SO interacted with the transcription factor FUBP1 and the NIPA1 gene. The effect of NIPA1-SO on NIPA1 protein levels was reversed by the knockdown of FUBP1. NIPA1-SO overexpression increased, whilst NIPA1-SO knockdown decreased BMPR2 levels; these effects were enhanced by the knockdown of NIPA1. The overexpression of NIPA1-SO reduced while NIPA1-SO knockdown increased monocyte adhesion to HUVECs; these effects were diminished by the knockdown of BMPR2. The lentivirus-mediated-overexpression of NIPA1-SO or gene-targeted knockout of NIPA1 in low-density lipoprotein receptor-deficient mice reduced monocyte-endothelium adhesion and atherosclerotic lesion formation. Collectively, these findings revealed a novel anti-atherosclerotic role for the lncRNA NIPA1-SO and highlighted its inhibitory effects on vascular inflammation and intracellular cholesterol accumulation by binding to FUBP1 and consequently repressing NIPA1 expression.

Chronic Kidney Disease Is Characterized by Expansion of a Distinct Proinflammatory Intermediate Monocyte Subtype and by Increased Monocyte Adhesion to Endothelial Cells.

CKD is accompanied by abnormal inflammation, which contributes to progressive loss of functional renal tissue and accelerated cardiovascular disease. Although studies have documented that dysregulation of monocyte maturation and function is associated with CKD and its complications, it is not well characterized. This study reveals that a distinctive human monocyte subtype with high propensity for releasing proinflammatory mediators and activating endothelial cells is increased in adults with CKD compared with adults with high cardiovascular risk and normal kidney function. It also demonstrates that human monocyte adhesion to endothelial layers and responses to specific inflammatory migration signals are enhanced in CKD. These findings offer insights into the mechanisms of CKD-associated intravascular and localized inflammation and may suggest potential targets for therapeutic interventions. Cardiovascular disease (CVD) in patients with CKD is associated with increased circulating intermediate monocytes (IMs). Dysregulation of monocyte maturation and function is associated with CKD and its complications, but it is incompletely characterized. To explore monocyte repertoire abnormalities in CKD, we studied properties of monocyte subpopulations, including IM subpopulations distinguished by HLA-DR expression level, in individuals with or without CKD. Using flow cytometry, we profiled monocyte populations in blood samples from adults with CKD, healthy volunteers (HVs), and patient controls (PCs) with high CVD risk. Monocyte subpopulations were also derived from single-cell RNA-sequencing profiles of paired blood and biopsy samples from kidney transplant recipients. We quantified intracellular cytokine production, migration, and endothelial adhesion in ex vivo assays of PBMCs. Of four predefined blood monocyte subpopulations, only HLA-DR hi IMs were increased in individuals with CKD compared with HVs and PCs. In HVs and patients with CKD, LPS-stimulated HLA-DR hi IMs isolated from blood produced higher amounts of TNF and IL-1 β than other monocyte populations. Single-cell analysis revealed four monocyte clusters common to blood and kidneys, including an HLA-DR hi IM-like cluster that was enriched in kidneys versus blood. Migration toward CCL5 and CX3CL1 and adhesion to primary endothelial cell layers were increased in monocyte subpopulations in individuals with CKD compared with HVs. Monocyte adhesion to endothelial cells was partly dependent on CX3CR1/CX3CL1 interaction. CKD is associated with an increased number of a distinctive proinflammatory IM subpopulation and abnormalities of monocyte migration and endothelial adhesion. Dysregulated monocyte maturation and function may represent targetable factors contributing to accelerated CVD in CKD.

Whole-Transcriptome Sequencing Analyses of Nuclear Antixoxidant-1 in Endothelial Cells: Role in Inflammation and Atherosclerosis.

Inflammation, oxidative stress, and copper (Cu) play an important role in cardiovascular disease, including atherosclerosis. We previously reported that cytosolic Cu chaperone antioxidant-1 (Atox1) translocates to the nucleus in response to inflammatory cytokines or exogenous Cu and that Atox1 is localized at the nucleus in the endothelium of inflamed atherosclerotic aorta. However, the roles of nuclear Atox1 and their function are poorly understood. Here we showed that Atox1 deficiency in ApoE−/− mice with a Western diet exhibited a significant reduction of atherosclerotic lesion formation. In vitro, adenovirus-mediated overexpression of nuclear-targeted Atox1 (Ad-Atox1-NLS) in cultured human endothelial cells (ECs) increased monocyte adhesion and reactive oxygen species (ROS) production compared to control cells (Ad-null). To address the underlying mechanisms, we performed genome-wide mapping of Atox1-regulated targets in ECs, using an unbiased systemic approach integrating sequencing data. Combination of ChIP-Seq and RNA-Seq analyses in ECs transfected with Ad-Atox1-NLS or Ad-null identified 1387 differentially expressed genes (DEG). Motif enrichment assay and KEGG pathway enrichment analysis revealed that 248 differentially expressed genes, including inflammatory and angiogenic genes, were regulated by Atox1-NLS, which was then confirmed by real-time qPCR. Among these genes, functional analysis of inflammatory responses identified CD137, CSF1, and IL5RA as new nuclear Atox1-targeted inflammatory genes, while CD137 is also a key regulator of Atox1-NLS-induced ROS production. These findings uncover new nuclear Atox1 downstream targets involved in inflammation and ROS production and provide insights into the nuclear Atox1 as a potential therapeutic target for the treatment of inflammatory diseases such as atherosclerosis.

The Acid Sphingomyelinase Inhibitor Amitriptyline Ameliorates TNF-α-Induced Endothelial Dysfunction.

Inflammation associated endothelial cell (EC) dysfunction is key to atherosclerotic disease. Recent studies have demonstrated a protective role of amitriptyline in cardiomyocytes induced by hypoxia/reoxygenation. However, the mechanism by which amitriptyline regulates the inflammatory reaction in ECs remains unknown. Thus, the aim of this study was to investigate whether amitriptyline protects against inflammation in TNF-α-treated ECs. HUVECs were incubated with amitriptyline (2.5 μM) or TNF-α (20 ng/ml) for 24 h. EdU, tube formation, transwell, DHE fluorescence staining, and monocyte adhesion assays were performed to investigate endothelial function. Thoracic aortas were isolated from mice, and vascular tone was measured with a wire myograph system. The levels of ICAM-1, VCAM-1, MCP-1, phosphorylated MAPK and NF-κB were detected using western blotting. Amitriptyline increased the phosphorylation of nitric oxide synthase (eNOS) and the release of NO. Amitriptyline significantly inhibited TNF-α-induced increases in ASMase activity and the release of ceramide and downregulated TNF-α-induced expression of proinflammatory proteins, including ICAM-1, VCAM-1, and MCP-1 in ECs, as well as the secretion of sICAM-1 and sVCAM-1. TNF-α treatment obviously increased monocyte adhesion and ROS production and impaired HUVEC proliferation, migration and tube formation, while amitriptyline rescued proliferation, migration, and tube formation and decreased monocyte adhesion and ROS production. Additionally, we demonstrated that amitriptyline suppressed TNF-α-induced MAPK phosphorylation as well as the activity of NF-κB in HUVECs. The results showed that the relaxation response of aortic rings to acetylcholine in the WT-TNF-α group was much lower than that in the WT group, and the sensitivity of aortic rings to acetylcholine in the WT-TNF-α group and WT-AMI-TNF-α group was significantly higher than that in the WT-TNF-α group. These results suggest that amitriptyline reduces endothelial inflammation, consequently improving vascular endothelial function. Thus, the identification of amitriptyline as a potential strategy to improve endothelial function is important for preventing vascular diseases.

Ractopamine at legal residue dosage accelerates atherosclerosis by inducing endothelial dysfunction and promoting macrophage foam cell formation

Ractopamine, a synthetic β-adrenoreceptor agonist, is used as an animal feed additive to increase food conversion efficiency and accelerate lean mass accretion in farmed animals. The U.S. Food and Drug Administration claimed that ingesting products containing ractopamine residues at legal dosages might not cause short-term harm to human health. However, the effect of ractopamine on chronic inflammatory diseases and atherosclerosis is unclear. Therefore, we investigated the effects of ractopamine on atherosclerosis and its action mechanism in apolipoprotein E-null (apoe−/−) mice and human endothelial cells (ECs) and macrophages. Daily treatment with ractopamine for four weeks increased the body weight and the weight of brown adipose tissues and gastrocnemius muscles. However, it decreased the weight of white adipose tissues in apoe−/− mice. Additionally, ractopamine exacerbated hyperlipidemia and systemic inflammation, deregulated aortic cholesterol metabolism and inflammation, and accelerated atherosclerosis. In ECs, ractopamine treatment induced endothelial dysfunction and increased monocyte adhesion and transmigration across ECs. In macrophages, ractopamine dysregulated cholesterol metabolism by increasing oxidized low-density lipoprotein (oxLDL) internalization and decreasing reverse cholesterol transporters, increasing oxLDL-induced lipid accumulation. Collectively, our findings revealed that ractopamine induces EC dysfunction and deregulated cholesterol metabolism of macrophages, which ultimately accelerates atherosclerosis progression.

Isobavachalcone attenuates TNF-α-induced ICAM-1 and VCAM-1 expression in human umbilical vein endothelial cells by regulating the NF-κB signaling pathway

Vascular inflammation activated by pro-inflammatory cytokines is an inflammatory response that occurs in the early stages of atherosclerosis. Endothelial dysfunction in vascular inflammation begins with the expression of cell surface adhesion molecules by pro-inflammatory cytokines. The purpose of this study was to evaluate and verify the vascular inflammatory effects of isobavachalcone. In this study, we investigated the effects of isobavachalcone on inflammatory responses in vascular inflammation induced by the tumor necrosis factor-α (TNF-α) in human umbilical vein endothelial cells (HUVECs). TNF-α stimulation significantly increased the expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) proteins, and concentration-dependently decreased by isobavachalcone in HUVECs. Isobavachalcone suppressed TNF-α-induced ICAM-1 and VCAM-1 expression in HUVECs, thereby inhibiting TNF-α-induced increase in monocyte adhesion. In addition, isobavachalcone decreased the phosphorylation of the NF-κB (necrosis factor κB) p65 subunit. The findings of this study demonstrate that isobavachalcone prevents TNF-α-induced vascular inflammation and has the potential to protect against the early progression of atherosclerosis.

FVIII regulates the molecular profile of endothelial cells: functional impact on the blood barrier and macrophage behavior.

Hemophilia A is an inherited X-linked recessive bleeding disorder caused by deficient activity of blood coagulation factor VIII (FVIII). In addition, hemophilia patients show associated diseases including osteopenia, altered inflammation and vascular fragility which may represent the consequence of recurrent bleeding or may be related to the direct FVIII deficiency. Nowadays, recombinant FVIII is proposed to treat hemophilia patients with no circulating FVIII inhibitor. Initially described as a coenzyme to factor IXa for initiating thrombin generation, there is emerging evidence that FVIII is involved in multiple biological systems, including bone, vascular and immune systems. The present study investigated: (i) the functional activities of recombinant human FVIII (rFVIII) on endothelial cells, and (ii) the impact of rFVIII activities on the functional interactions of human monocytes and endothelial cells. We then investigated whether rFVIII had a direct effect on the adhesion of monocytes to the endothelium under physiological flow conditions. We observed that direct biological activities for rFVIII in endothelial cells were characterized by: (i) a decrease in endothelial cell adhesion to the underlying extracellular matrix; (ii) regulation of the transcriptomic and protein profiles of endothelial cells; (iii) an increase in the vascular tubes formed and vascular permeability in vitro; and (iv) an increase in monocyte adhesion activated endothelium and transendothelial migration. By regulating vascular permeability plus leukocyte adhesion and transendothelial migration, the present work highlights new biological functions for FVIII.

A human pluripotent stem cell-based model of SARS-CoV-2 infection reveals an ACE2-independent inflammatory activation of vascular endothelial cells through TLR4.

SummaryTo date, the direct causative mechanism of SARS-CoV-2-induced endotheliitis remains unclear. Here, we report that human ECs barely express surface ACE2, and ECs express less intracellular ACE2 than non-ECs of the lungs. We ectopically expressed ACE2 in hESC-ECs to model SARS-CoV-2 infection. ACE2-deficient ECs are resistant to the infection but are more activated than ACE2-expressing ones. The virus directly induces endothelial activation by increasing monocyte adhesion, NO production, and enhanced phosphorylation of p38 mitogen-associated protein kinase (MAPK), NF-κB, and eNOS in ACE2-expressing and -deficient ECs. ACE2-deficient ECs respond to SARS-CoV-2 through TLR4 as treatment with its antagonist inhibits p38 MAPK/NF-κB/ interleukin-1β (IL-1β) activation after viral exposure. Genome-wide, single-cell RNA-seq analyses further confirm activation of the TLR4/MAPK14/RELA/IL-1β axis in circulating ECs of mild and severe COVID-19 patients. Circulating ECs could serve as biomarkers for indicating patients with endotheliitis. Together, our findings support a direct role for SARS-CoV-2 in mediating endothelial inflammation in an ACE2-dependent or -independent manner.