Protective effects of curcumin on corneal endothelial cell PANoptosis and monocyte adhesion induced by tumor necrosis factor-alpha and interferon-gamma in rats.

Decrease of human corneal endothelial cell (CEC) density leads to corneal edema, progressive corneal opacity, and reduced visual acuity. A reduction in CEC density may be related to elevated levels of inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interferon (INF)-γ. PANoptosis, characterized by the activation of apoptosis, necroptosis, and pyroptosis, could be a factor in the loss of CECs driven by TNF-α and INF-γ. Cytokines also stimulate monocytes adhesion to endothelium. It has been shown in previous research that curcumin plays protective roles against numerous corneal inflammatory diseases. However, it is not determined whether curcumin acts as an anti-PANoptotic agent or if it mitigates monocyte adhesion to CECs. Therefore, this research aimed to explor the potential therapeutic effects of curcumin and its underlying mechanisms in the loss of CECs. CEC injury models were established, and curcumin was injected subconjunctivally. Clinical evaluation of the corneas was conducted using a scoring system and anterior segment photography. Corneal observation was performed with hematoxylin and eosin staining and immunostaining of zona occludens-1(ZO-1). Apoptotic cells within the corneal endothelium were observed using TUNEL staining. The detection of primary proteins expression was accomplished through Western blot analysis. Interleukin (IL)-1ß and macrophage chemotactic protein 1 (MCP-1) levels were determined via ELISA, while the expression of cleaved caspase-3, gasdermin-D (GSDMD), phosphor-mixed lineage kinase domain-like protein (p-MLKL) and intercellular cell adhesion molecule-1 were confirmed by immunofluorescence. Myeloperoxidase (MPO) activity was measured in aqueous humors. Curcumin treatment attenuated the loss of CECs and corneal edema caused by TNF-α and IFN-γ. Besides, it decreased the count of TUNEL-positive cells, and inhibited the upregulation of cleaved caspase-3, cleaved caspase-6, cleaved caspase-7, and cleaved poly (ADP-ribose) polymerase. Moreover, both the expression and phosphorylation of MLKL and receptor-interacting protein 3 were decreased in curcumin-treated rats. Furthermore, curcumin also lowered the expression of cleaved caspase-1, diminished the levels of IL1ß and MCP-1, and inhibited the activity of MPO. Besides, the expression of intercellular cell adhesion molecule-1, vascular cell adhesion molecule-1, as well as the number of CD11b-positive cells adhered to the CECs decreased for the administration of curcumin.

Anti-atherogenic mechanism of ethanol extract of Christia vespertilionis (L.f.) Bakh. F. Leaves in vitro.

BACKGROUND: Vascular inflammation is the key event in early atherogenesis. Pro-inflammatory endothelial cells induce monocyte recruitment into the sub-endothelial layer of the artery. This requires endothelial expression of adhesion molecules namely intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), alongside chemokines production. Christia vespertilionis (L.f.) Bakh.f. (CV) possesses anti-inflammatory property. However, its potential anti-atherogenic effect in the context of vascular inflammation has yet to be explored. PURPOSE: To evaluate the anti-atherogenic mechanism of 80% ethanol extract of CV leaves on tumor necrosis factor-α (TNF-α)-activated human umbilical vein endothelial cells (HUVECs). METHODS: Qualitative analysis of the CV extract was carried out by using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The cell viability of HUVECs treated with CV extract was determined by MTT assay. The effect of CV extract on monocyte adhesion was determined by monocyte-endothelial adhesion assay. Protein expressions of ICAM-1, VCAM-1 and nuclear factor-kappa B (NF-κB) signaling pathway were determined by western blot while production of monocyte chemoattractant protein-1 (MCP-1) was determined by ELISA. RESULTS: LC-MS/MS analysis showed that CV extract composed of five main compounds, including schaftoside, orientin, isovitexin, 6-caffeoyl-D-glucose, and 3,3'-di-O-methyl ellagic acid. Treatment of CV extract at a concentration range from 5 to 60 µg/mL for 24 h maintained HUVECs viability above 90 %, therefore concentrations of 20, 40 and 60 µg/mL were selected for the subsequent experiments. All concentrations of CV extract showed a significant inhibitory effect on monocyte adhesion to TNF-α-activated HUVECs (p < 0.05). In addition, the protein expressions of ICAM-1 and VCAM-1 were significantly attenuated by CV in a concentration dependent manner (p < 0.001). At all tested concentrations, CV extract also exhibited significant inhibition on the production of MCP-1 (p < 0.05). Moreover, CV extract significantly inhibited TNF-α-induced phosphorylation of inhibitor of nuclear factor-κB kinase alpha/beta (IKKα/ß), inhibitor kappa B-alpha (IκBα), NF-κB and nuclear translocation of NF-κB (p < 0.05). CONCLUSION: CV extract inhibited monocyte adhesion to endothelial cells by suppressing protein expressions of cell adhesion molecules and production of chemokines through downregulation of NF-κB signaling pathway. Thus, CV has the potential to be developed as an anti-atherogenic agent for early treatment of atherosclerosis.

Actinidia arguta Extract Containing Myo-Inositol Suppresses TNF-α-Induced VCAM-1 Expression and Monocyte Adhesion to Endothelial Cells via Inhibition of the PTEN/Akt/GSK-3ß and NF-κB Signaling Pathways.

The primary inflammatory process in atherosclerosis, a major contributor to cardiovascular disease, begins with monocyte adhering to vascular endothelial cells. Actinidia arguta (kiwiberry) is an edible fruit that contains various bioactive components. While A. arguta extract (AAE) has been recognized for its anti-inflammatory characteristics, its specific inhibitory effect on early atherogenic events has not been clarified. We used tumor necrosis factor-α (TNF-α)-stimulated human umbilical vein endothelial cells (HUVECs) for an in vitro model. AAE effectively hindered the attachment of THP-1 monocytes and reduced the expression of vascular cell adhesion molecule-1 (VCAM-1) in HUVECs. Transcriptome analysis revealed that AAE treatment upregulated phosphatase and tensin homolog (PTEN), subsequently inhibiting phosphorylation of AKT and glycogen synthase kinase 3ß (GSK3ß) in HUVECs. AAE further hindered phosphorylation of AKT downstream of the nuclear factor kappa B (NF-κB) signaling pathway, leading to suppression of target gene expression. Oral administration of AAE suppressed TNF-α-stimulated VCAM-1 expression, monocyte-derived macrophage infiltration, and proinflammatory cytokine expression in C57BL/6 mouse aortas. Myo-inositol, identified as the major compound in AAE, played a key role in suppressing THP-1 monocyte adhesion in HUVECs. These findings suggest that AAE could serve as a nutraceutical for preventing atherosclerosis by inhibiting its initial pathogenesis.

TRIM65 Suppresses oxLDL-induced Endothelial Inflammation by Interaction with VCAM-1 in Atherogenesis.

BACKGROUND AND OBJECTIVE: Endothelial cell activation, characterized by increased levels of vascular cell adhesion molecule 1 (VCAM-1), plays a crucial role in the development of atherosclerosis (AS). Therefore, inhibition of VCAM-1-mediated inflammatory response is of great significance in the prevention and treatment of AS. The tripartite motif (TRIM) protein-TRIM65 is involved in the regulation of cancer development, antivirals and inflammation. We aimed to study the functions of TRIM65 in regulating endothelial inflammation by interacting with VCAM-1 in atherogenesis. METHODS AND RESULTS: In vitro, we report that human umbilical vein endothelial cells (HUVECs) treated with oxidized low-density lipoprotein (oxLDL) significantly upregulate the expression of TRIM65 in a time- and dose-dependent manner. Overexpression of TRIM65 reduces oxLDL-triggered VCAM-1 protein expression, decreases monocyte adhesion to HUVECs and inhibits the production of the inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α as well as endothelial oxLDL transcytosis. In contrast, siRNA-mediated knockdown of TRIM65 promotes the expression of VCAM-1, resulting in increased adhesion of monocytes and the release of the inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α and enhances endothelial oxLDL transcytosis. In vivo, we measured the high expression of TRIM65 in ApoE-/- mouse aortic plaques compared to C57BL/6J mouse aortic plaques. Then, we examined whether the blood levels of VCAM-1 were higher in TRIM65 knockout ApoE-/- mice than in control mice induced by a Western diet. Furthermore, Western blot results showed that the protein expression of VCAM-1 was markedly enhanced in TRIM65 knockout ApoE-/- mouse aortic tissues compared to that of the controls. Immunofluorescence staining revealed that the expression of VCAM-1 was significantly increased in atherosclerotic plaques of TRIM65-/-/ApoE-/- aortic vessels compared to ApoE-/- controls. Mechanistically, TRIM65 specifically interacts with VCAM-1 and targets it for K48-linked ubiquitination. CONCLUSION: Our studies indicate that TRIM65 attenuates the endothelial inflammatory response by targeting VCAM-1 for ubiquitination and provides a potential therapeutic target for the inhibition of endothelial inflammation in AS.

In Vitro Anti-Inflammatory and Vasculoprotective Effects of Red Cell Extract from the Black Sea Urchin Arbacia lixula.

Sea urchins have emerged as an important source of bioactive compounds with anti-inflammatory and antioxidant properties relevant to human health. Since inflammation is a crucial pathogenic process in the development and progression of atherosclerosis, we here assessed the potential anti-inflammatory and vasculoprotective effects of coelomic red-cell methanolic extract of the black sea urchin Arbacia lixula in an in vitro model of endothelial cell dysfunction. Human microvascular endothelial cells (HMEC-1) were pretreated with A. lixula red-cell extract (10 and 100 µg/mL) before exposure to the pro-inflammatory cytokine tumor necrosis factor (TNF)-α. The extract was non-toxic after 24 h cell treatment and was characterized by antioxidant power and phenol content. The TNF-α-stimulated expression of adhesion molecules (VCAM-1, ICAM-1) and cytokines/chemokines (MCP-1, CCL-5, IL-6, IL-8, M-CSF) was significantly attenuated by A. lixula red-cell extract. This was functionally accompanied by a reduction in monocyte adhesion and chemotaxis towards activated endothelial cells. At the molecular level, the tested extract significantly counteracted the TNF-α-stimulated activation of the pro-inflammatory transcription factor NF-κB. These results provide evidence of potential anti-atherosclerotic properties of A. lixula red-cell extract, and open avenues in the discovery and development of dietary supplements and/or drugs for the prevention or treatment of cardiovascular diseases.

Comparative study of the effects of cigarette smoke versus next-generation tobacco and nicotine product extracts on inflammatory biomarkers of human monocytes.

Monocytes exhibiting a pro-inflammatory phenotype play a key role in adhesion and development of atherosclerotic plaques. As an alternative to smoking, next-generation tobacco and nicotine products (NGP) are now widely used. However, little is known about their pro-inflammatory effects on monocytes. We investigated cell viability, anti-oxidant and pro-inflammatory gene and protein expression in THP-1 monocytes after exposure to aqueous smoke extracts (AqE) of a heated tobacco product (HTP), an electronic cigarette (e-cig), a conventional cigarette (3R4F) and pure nicotine (nic). Treatment with 3R4F reduced cell viability in a dose-dependent manner, whereas exposure to alternative smoking products showed no difference to control. At the highest non-lethal dose of 3R4F (20%), the following notable mRNA expression changes were observed for 3R4F, HTP, and e-cig respectively, relative to control; HMOX1 (6-fold, < 2-fold, < 2-fold), NQO1 (3.5-fold, < 2-fold, < 2-fold), CCL2 (4-fold, 3.5-fold, 2.5-fold), IL1B (4-fold, 3-fold, < 2-fold), IL8 (5-fold, 2-fold, 2-fold), TNF (2-fold, 2-fold, < 2-fold) and ICAM1 was below the 2-fold threshold for all products. With respect to protein expression, IL1B (3-fold, < 2-fold, < 2-fold) and IL8 (3.5-fold, 2-fold, 2-fold) were elevated over the 2-fold threshold, whereas CCL2, TNF, and ICAM1 were below 2-fold expression for all products. At higher doses, greater inductions were observed with all extracts; however, NGP responses were typically lower than 3R4F. In conclusion, anti-oxidative and pro-inflammatory processes were activated by all products. NGPs overall showed lower responses relative to controls than THP-1 cells exposed to 3R4F AqE.

Human vasculature-on-a-chip with macrophage-mediated endothelial activation: The biological effect of aerosol from heated tobacco products on monocyte adhesion.

Heated tobacco products (HTPs) are expected to have the potential to reduce risks of smoking-associated cardiovascular disease (CVD). However, mechanism-based investigations of the effect of HTPs on atherosclerosis remain insufficient and further studies under human-relevant situations are desired for deeper understanding of the reduced risk potential of HTPs. In this study, we first developed an in vitro model of monocyte adhesion by considering macrophage-derived proinflammatory cytokine-mediated endothelial activation using an organ-on-a-chip (OoC), which provided great opportunities to mimic major aspects of human physiology. Then biological activities of aerosol from three different types of HTPs in terms of monocyte adhesion were compared with that of cigarette smoke (CS). Our model showed that the effective concentration ranges of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were close to the actual condition in CVD pathogenesis. The model also showed that monocyte adhesion was less induced by each HTP aerosol than CS, which may be caused by less proinflammatory cytokine secretion. In summary, our vasculature-on-a-chip model assessed the difference in biological effects between cigarettes and HTPs, and suggested a reduced risk potential of HTPs for atherosclerosis.

The Endothelial Glycocalyx as a Target of Excess Soluble Fms-like Tyrosine Kinase-1.

Soluble fms-like tyrosine kinase-1 (sFlt-1) is a secreted protein that binds heparan sulfate expressed on the endothelial glycocalyx (eGC). In this paper we analyze how excess sFlt-1 causes conformational changes in the eGC, leading to monocyte adhesion, a key event triggering vascular dysfunction. In vitro exposure of primary human umbilical vein endothelial cells to excess sFlt-1 decreased eGC height and increased stiffness as determined by atomic force microscopy (AFM). Yet, structural loss of the eGC components was not observed, as indicated by Ulex europaeus agglutinin I and wheat germ agglutinin staining. Moreover, the conformation observed under excess sFlt-1, a collapsed eGC, is flat and stiff with unchanged coverage and sustained content. Functionally, this conformation increased the endothelial adhesiveness to THP-1 monocytes by about 35%. Heparin blocked all these effects, but the vascular endothelial growth factor did not. In vivo administration of sFlt-1 in mice also resulted in the collapse of the eGC in isolated aorta analyzed ex vivo by AFM. Our findings show that excess sFlt-1 causes the collapse of the eGC and favors leukocyte adhesion. This study provides an additional mechanism of action by which sFlt-1 may cause endothelial dysfunction and injury.

TRIM59 attenuates ox-LDL-induced endothelial cell inflammation, apoptosis, and monocyte adhesion through AnxA2.

Background: Atherosclerosis (AS), a chronic inflammatory vascular disease, is a cause of heart attack and ischemic stroke. Tripartite motif-containing protein 59 (TRIM59), a member of the tripartite motif family, has been reported to be involved in inflammatory diseases. This study was to investigate the role of TRIM59 in oxidized low-density lipoprotein (ox-LDL)-induced endothelial cells and examine the mechanism of TRIM59. Methods: To simulate a cellular model of AS in vitro, varying concentrations of ox-LDL (i.e., 20, 40, 60, 80, and 100 µg/mL) were used to treat the human umbilical vein endothelial cells (HUVECs) for 24 h. The messenger ribonucleic acid (RNA) and protein levels of TRIM59, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), and annexin 2 (AnxA2) were examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. The transfection efficacy of overexpression (Ov)-TRIM59 and small-interfering RNA-AnxA2 was examined by RT-qPCR and western blot. Cell counting kit-8 assays, lactate dehydrogenase (LDH) assays, enzyme-linked immunosorbent assays, and terminal-deoxynucleotidyl transferase mediated nick end labeling staining were used to examine viability, LDH expression, inflammation, and apoptosis in HUVECs. The protein levels of B-cell lymphoma 2, Bcl-2-associated X (BAX), cleaved caspase3, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 were assessed by western blot. Additionally, the adhesion of THP-1 to ox-LDL-induced HUVECs was detected using monocyte adhesion assays and the binding of TRIM59 and AnxA2 was verified by co-immunoprecipitation. Results: This study showed that TRIM59 expression was decreased in the ox-LDL-induced HUVECs while LOX-1 expression was increased. After transfection with Ov-TRIM59, TRIM59 in ox-LDL-induced HUVECs was increased, and TRIM59 overexpression alleviated the viability damage, inflammation, and apoptosis of the ox-LDL-induced HUVECs. In addition, THP-1 adhesion to the ox-LDL-induced HUVECs was also suppressed by TRIM59 overexpression. This study also showed that TRIM59 could bind to AnxA2 and promote AnxA2 expression in ox-LDL-stimulated HUVECs. Moreover, the rescue experiments revealed that TRIM59 suppressed the viability damage, inflammation, apoptosis, and monocyte adhesion of the ox-LDL-induced HUVECs via AnxA2. Conclusions: TRIM59 protected against ox-LDL-induced AS by binding to AnxA2.

Integrin CD11b Contributes to Hypertension and Vascular Dysfunction Through Mediating Macrophage Adhesion and Migration.

BACKGROUND: Leukocyte adhesion to endothelium is an early inflammatory response and is mainly controlled by the ß2-integrins. However, the role of integrin CD11b/CD18 in the pathogenesis of hypertension and vascular dysfunction is unclear. METHODS: Hypertension was established by angiotensin II (490 ng/kg·per min) or deoxycorticosterone acetate salt. Hypertensive responses were studied in CD11b-deficient (CD11b-/-) mice, bone marrow transplanted and wild-type (WT) mice that were administered anti-CD11b neutralizing antibody or agonist leukadherin-1. Blood pressure was monitored with tail-cuff method and radiotelemetry. Blood and vascular inflammatory cells were assessed by flow cytometry. Aortic remodeling and function were examined using histology and aortic ring analysis. Cell adhesion and migration were evaluated in vitro. The relationship between circulating CD11b+ immune cells and hypertension was analyzed in patients with hypertension. RESULTS: We found that CD11b and CD18 expression as well as the CD45+CD11b+CD18+ myeloid cells were highly increased in the aorta of angiotensin II-infused mice. Ablation or pharmacological inhibition of CD11b in mice significantly alleviated hypertension, aortic remodeling, superoxide generation, vascular dysfunction, and the infiltration of CD11b+ macrophages through reducing macrophage adhesion and migration. These effects were confirmed in WT mice reconstituted with CD11b-deficient bone marrow cells. Conversely, angiotensin II-induced hypertensive response was exacerbated by CD11b agonist leukadherin-1. Notably, circulating CD45+CD11b+CD18+ myeloid cells and the ligand levels in hypertensive patients were significantly higher than in normotensive controls. CONCLUSIONS: We demonstrated a critical significance of CD11b+ myeloid cells in hypertension and vascular dysfunction. Targeting CD11b may represent a novel therapeutic option for hypertension.

Persicaria minor (Huds.) Opiz Prevents In Vitro Atherogenesis by Attenuating Tumor Necrosis Factor-α-Induced Monocyte Adhesion to Human Umbilical Vein Endothelial Cells.

Persicaria minor (Huds.) Opiz is an herb with anti-inflammatory, antioxidant, and anti-atherosclerosis effects. Nevertheless, the mechanism underlying its anti-atherosclerosis effect is poorly comprehended. This in vitro study assessed the protective effects of standardized aqueous extract of P. minor leaves (PM) on tumor necrosis factor-α (TNF-α)-induced monocyte adhesion to human umbilical vein endothelial cells (HUVEC), which is one of the pivotal early steps in atherogenesis. The results showed that PM decreased the mRNA and protein expression of cellular adhesion molecules, vascular adhesion molecule-1 and intercellular adhesion molecule-1, resulting in reduced adhesion of monocytes to HUVEC. Additionally, PM inhibited nuclear factor kappaB (NF-κB) activation as indicated by reduced NF-κB p65 levels in TNF-α-induced HUVEC. Overall, PM could prevent in vitro atherogenesis by inhibiting NF-κB activation and adhesion of monocytes to HUVEC. The effects of PM are probably mediated by its bioactive compound, quercetin-3-O-glucuronide. The findings may provide a rationale for the in vivo anti-atherosclerosis effect of PM, and support its potential use in atherosclerosis.

CORM-2 prevents human gingival fibroblasts from lipoteichoic acid-induced VCAM-1 and ICAM-1 expression by inhibiting TLR2/MyD88/TRAF6/PI3K/Akt/ROS/NF-κB signaling pathway.

Periodontal diseases are prevalent worldwide. Lipoteichoic acid (LTA), a major component of gram-positive bacteria, may play a key role in periodontally inflammatory diseases. Carbon monoxide (CO) is a critical messenger in many biological processes. It can elicit various biological properties, especially anti-inflammatory effects. As the straight administration of CO remains difficult, CO-releasing molecules (CO-RMs) are emerging as promising alternatives. To explore the pharmacological actions and signaling pathways of CO battling LTA-induced periodontal inflammation, this study investigated the cytoprotective effects of CORM-2 against the adhesion of THP-1 monocytes to human gingival fibroblasts (HGFs) and the underlying molecular mechanism. After exposing HGFs to LTA with or without CORM-2 pretreatment, monocyte adhesion was determined. VCAM-1 and ICAM-1 expression in HGFs was measured by real-time PCR. To identify the signaling pathways of CO involved in the cytoprotective effects of CORM-2, HGFs underwent pharmacological or genetical interventions before LTA incubation. The expression and/or activity of possible regulatory molecules were determined. The release of pro-inflammatory cytokines, including IL-1ß, IL-6, and TNF-α, were measured using ELISA. The results showed that LTA increased cytokine production and upregulated VCAM-1 and ICAM-1 expression in HGFs, promoting monocyte adhesion. These events were dependent on TLR2/MyD88/TRAF6- and PI3K/Akt/NADPH oxidase/ROS-regulated NF-κB activation. CORM-2 inhibited LTA-induced inflammatory cascades in HGFs, in which CO seemed to be the hitman. To conclude, CO released from CORM-2 can prevent the LTA-stimulated HGFs from increasing VCAM-1 and ICAM-1 expression and promoting monocyte adhesion by inhibiting TLR2/MyD88/TRAF6 association and PI3K/Akt/NADPH oxidase/ROS signaling, both converge on the canonical NF-κB activation.

Quercetin Mitigates Endothelial Activation in a Novel Intestinal-Endothelial-Monocyte/Macrophage Coculture Setup.

Atherosclerosis initiation is associated with a pro-inflammatory state of the endothelium. Quercetin is a flavonoid abundantly present in plant-based foods, with a possible impact on cardiovascular health. In this study, the effects of quercetin on lipopolysaccharide (LPS)-mediated endothelial inflammation and monocyte adhesion and migration, which are initial steps of the atherogenic process, are studied. Novel in vitro multicellular models simulating the intestinal-endothelial-monocytes/macrophages axis allowed to combine relevant intestinal flavonoid absorption, metabolism and efflux, and the consequent bioactivity towards peripheral endothelial cells. In this triple coculture, quercetin exposure decreased monocyte adhesion to and macrophage migration through an LPS-stressed endothelium, and this was associated with significantly lower levels of soluble vascular cell adhesion molecule-1 (sVCAM-1). Furthermore, quercetin decreased the pro-inflammatory cell environment upon LPS-induced endothelial activation, in terms of tumor necrosis factor- α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), and sVCAM-1 expression. These findings highlight a mode-of-action by which quercetin may positively impact the initial states of atherosclerosis under more physiologically relevant conditions in terms of quercetin concentrations, metabolites, and intercellular crosstalk.

3-(3-Hydroxyphenyl)propionic acid, a microbial metabolite of quercetin, inhibits monocyte binding to endothelial cells via modulating E-selectin expression.

Adhesion of monocytes to endothelial cells is an important initiating step in atherogenesis. One of the most abundant flavonoids in the diet, quercetin has been reported to inhibit monocyte adhesion to endothelial cells. However, it is poorly absorbed in the upper gastrointestinal tract during oral intake but rather is metabolized by the intestinal microbiota into various phenolic acids. Since the biological properties of the microbial metabolites of quercetin remain largely unknown, herein, we investigated how the microbial metabolite of quercetin, 3-(3-hydroxyphenyl)propionic acid (3HPPA) impact monocyte adhesion to endothelial cells. Direct treatment with 3HPPA for 24 h was not cytotoxic to human aortic endothelial cells (HAECs). Cotreatment with 3HPPA inhibited tumor necrosis factor α (TNFα)-induced adhesion of THP-1 monocytes to HAECs, and suppressed the upregulation of cell adhesion molecule E-selectin but not intercellular adhesion molecule 1 or vascular cell adhesion molecule 1. Furthermore, 3HPPA was found to inhibit TNFα-induced nuclear translocation and phosphorylation of the p65 subunit of nuclear factor κB (NF-κB). We conclude that 3HPPA mitigates the adhesion of monocytes to endothelial cells by suppressing the expression of the cell adhesion molecule E-selectin in HAECs via inhibition of the NF-κB pathway, providing additional evidence for the health benefits of dietary flavonoids and their microbial metabolites as therapeutic agents in atherosclerosis.

Natural Compound Resveratrol Attenuates TNF-Alpha-Induced Vascular Dysfunction in Mice and Human Endothelial Cells: The Involvement of the NF-κB Signaling Pathway.

Resveratrol, a natural compound in grapes and red wine, has drawn attention due to potential cardiovascular-related health benefits. However, its effect on vascular inflammation at physiologically achievable concentrations is largely unknown. In this study, resveratrol in concentrations as low as 1 µm suppressed TNF-α-induced monocyte adhesion to human EA.hy926 endothelial cells (ECs), a key event in the initiation and development of atherosclerosis. Low concentrations of resveratrol (0.25-2 µm) also significantly attenuated TNF-α-stimulated mRNA expressions of MCP-1/CCL2 and ICAM-1, which are vital mediators of EC-monocyte adhesion molecules and cytokines for cardiovascular plaque formation. Additionally, resveratrol diminished TNF-α-induced IκB-α degradation and subsequent nuclear translocation of NF-κB p65 in ECs. In the animal study, resveratrol supplementation in diet significantly diminished TNF-α-induced increases in circulating levels of adhesion molecules and cytokines, monocyte adhesion to mouse aortic ECs, F4/80-positive macrophages and VCAM-1 expression in mice aortas and restored the disruption in aortic elastin fiber caused by TNF-α treatment. The animal study also confirmed that resveratrol blocks the activation of NF-κB In Vivo. In conclusion, resveratrol at physiologically achievable concentrations displayed protective effects against TNF-α-induced vascular endothelial inflammation in vitro and In Vivo. The ability of resveratrol in reducing inflammation may be associated with its role as a down-regulator of the NF-κB pathway.

Comparative study of the effects of cigarette smoke versus next generation tobacco and nicotine product extracts on endothelial function.

Tobacco smoking and hemodynamic forces are key stimuli for the development of endothelial dysfunction. As an alternative to smoking, next generation tobacco and nicotine products (NGP) are now widely used. However, little is known about their potential pro-inflammatory and atherogenic effects on the endothelium. In this study, we analyzed key parameters of endothelial function after exposure to aqueous smoke extracts (AqE) of a heated tobacco product (HTP), an electronic cigarette (e-cig), a conventional cigarette (3R4F) and pure nicotine. All experiments were performed under atheroprotective high laminar or atherogenic low flow with primary human endothelial cells. Treatment with 3R4F, but not alternative smoking products, reduced endothelial cell viability and wound healing capability via the PI3K/AKT/eNOS(NOS3) pathway. Laminar flow delayed detrimental effects on cell viability by 3R4F treatment. 3R4F stimulation led to activation of NRF2 antioxidant defense system at nicotine concentrations ≥0.56 µg/ml and increased expression of its target genes HMOX1 and NQO1. Treatment with HTP revealed an induction of HMOX1 and NQO1 at dosages with ≥1.68 µg/ml nicotine, whereas e-cig and nicotine exposure had no impact. Analyses of pro-inflammatory genes revealed an increased ICAM1 expression under 3R4F treatment. 3R4F reduced VCAM1 expression in a dose-dependent manner; HTP treatment had similar but milder effects; e-cig and nicotine treatment had no impact. SELE expression was induced by 3R4F under static conditions. High laminar flow prevented this upregulation. Stimulation with laminar flow led to downregulation of CCL2 (MCP-1). From this downregulated level, only 3R4F increased CCL2 expression at higher concentrations. Finally, under static conditions, all components increased adhesion of monocytes to endothelial cells. Interestingly, only stimulation with 3R4F revealed increased monocyte adhesion under atherosclerosis-prone low flow. In conclusion, all product categories activated anti-oxidative or pro-inflammatory patterns. NGP responses were typically lower than in 3R4F exposed cells. Also, 3R4F stimulation led to an impaired endothelial wound healing and induced a pro-inflammatory phenotype compared to NGP treatment.

Interferons Are Pro-Inflammatory Cytokines in Sheared-Stressed Human Aortic Valve Endothelial Cells.

Calcific aortic valve disease (CAVD) is an athero-inflammatory process. Growing evidence supports the inflammation-driven calcification model, mediated by cytokines such as interferons (IFNs) and tumor necrosis factor (TNF)-α. Our goal was investigating IFNs' effects in human aortic valve endothelial cells (VEC) and the potential differences between aortic (aVEC) and ventricular (vVEC) side cells. The endothelial phenotype was analyzed by Western blot, qPCR, ELISA, monocyte adhesion, and migration assays. In mixed VEC populations, IFNs promoted the activation of signal transducers and activators of transcription-1 and nuclear factor-κB, and the subsequent up-regulation of pro-inflammatory molecules. Side-specific VEC were activated with IFN-γ and TNF-α in an orbital shaker flow system. TNF-α, but not IFN-γ, induced hypoxia-inducible factor (HIF)-1α stabilization or endothelial nitric oxide synthase downregulation. Additionally, IFN-γ inhibited TNF-α-induced migration of aVEC. Also, IFN-γ triggered cytokine secretion and adhesion molecule expression in aVEC and vVEC. Finally, aVEC were more prone to cytokine-mediated monocyte adhesion under multiaxial flow conditions as compared with uniaxial flow. In conclusion, IFNs promote inflammation and reduce TNF-α-mediated migration in human VEC. Moreover, monocyte adhesion was higher in inflamed aVEC sheared under multiaxial flow, which may be relevant to understanding the initial stages of CAVD.

Paeonia lactiflora Root Extract and Its Components Reduce Biomarkers of Early Atherosclerosis via Anti-Inflammatory and Antioxidant Effects In Vitro and In Vivo.

Although various physiological activities of compounds obtained from Paeonia lactiflora have been reported, the effects of P. lactiflora extract (PLE) on early atherosclerosis remain unclear. Therefore, in this study, we investigated the in vitro and in vivo antiatherosclerosis and in vitro antioxidant effects of PLE and its compounds. PLE suppresses the tumor necrosis factor (TNF)-α-induced capacity of THP-1 cells to adhere to human umbilical vein endothelial cells (HUVECs), vascular cell adhesion molecule (VCAM)-1 expression, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling in HUVECs. PLE also suppresses TNF-α-induced nuclear translocation of NF-κB p65 from cytosol as well as the enhanced TNFA and C-C motif chemokine ligand 2 (CCL2) mRNA expression in HUVECs. We identified and quantified the following PLE compounds using high-performance liquid chromatography with diode array detection: methyl gallate, oxypaeoniflorin, catechin, albiflorin, paeoniflorin, benzoic acid, benzoylpaeoniflorin, and paeonol. Among these, methyl gallate had the strongest inhibitory effect on monocyte adherence to TNF-α-induced HUVECs and the VCAM-1 expression. Reverse transcriptase real-time quantitative polymerase chain reaction showed that PLE compounds had a dissimilar inhibition effect on TNF-α-induced mRNA expression levels of CCL2, TNFA, and IL6 in HUVECs. Except for paeonol, the compounds inhibited lipopolysaccharide (LPS)-induced reactive oxygen species production in RAW264.7 cells. In vivo, oral administration of PLE improved TNF-α-induced macrophage infiltration to the vascular endothelium and expression of VCAM-1, as well as IL6 and TNFA gene expression in the main artery of mice. PLE could be useful as a nutraceutical material against early atherosclerosis via the combined effects of its components.

MiR-520b inhibits endothelial activation by targeting NF-κB p65-VCAM1 axis.

MiR-520b belongs to the miR-373/520 family, is expressed only in human and nonhuman primates. Previous reports indicated that the expression of miR-520b was repressed in human atherosclerotic plaque tissue compared with healthy vessels. However, the role of miR-520b in coronary artery disease still remains to be uncovered. In this study, we demonstrated that endothelial cells (ECs) in human atherosclerotic plaques expressed miR-520b and aimed to elucidate the impact of miR-520b on EC activation and inflammatory response. To determine the potential targets of miR-520b, we performed RNA-seq analysis by transfecting miR-520b mimics in ECs. The quantitative real-time PCR (qPCR) validation suggested that miR-520b over-expression reduced pro-inflammatory gene expression (e.g. ICAM1, VCAM1, SELE) while the inhibition of miR-520b induced their expression. By combining bioinformatics prediction and functional assays, we identified that RELA (Nuclear Factor-κB (NF-κB) Transcription Factor P65) was a direct target of miR-520b. Moreover, miR-520b mimics attenuated monocyte adhesion and monocyte trans-endothelial migration (the initial steps of atherosclerotic formation) in response to lipopolysaccharides (LPS) stimulation. Re-expression of a non-miR-targetable version of p65 could rescue the reduced monocyte cell attachment, suggesting that this process is NF-κB p65 dependent. MiR-520b reduced the abundance of NF-κB p65 in cytoplasmic fractions without corresponding increase in nuclear fractions, indicating that this regulation is independent of p65 translocation process. MiR-520b mimics attenuated the activity of VCAM-1 promoter, whereas miR-520b inhibitor activated its activity. However, miR-520b inhibitor had no effect on promoter activity containing the mutated NF-κB p65 binding sites, strongly demonstrating that the impact of miR-520b on VCAM1 gene is mediated by NF-κB p65. Thus, we concluded that miR-520b suppressed EC inflammation and the cross-talk between monocytes and ECs by down-regulating NF-κB p65-ICAM1/VCAM1 axis and might serve as a potential therapeutic target for EC dysfunction and atherosclerosis.

Gamma-glutamyl carboxylated Gas6 facilitates the prophylactic effect of vitamin K in inhibiting hyperlipidemia-associated inflammatory pathophysiology via arresting MCP-1/ICAM-1 mediated monocyte-hepatocyte adhesion.

Role of growth arrest-specific 6 (Gas6), member of vitamin K (VK)-dependent protein family in hyperlipidemia-associated inflammation remains unresolved. To address this, blood samples were collected from hyperlipidemic subjects and age-matched healthy controls and observed that gamma-glutamyl carboxylated Gas6 (Gla-Gas6) but not total Gas6 were significantly lower while pro-inflammatory markers, MCP-1 and ICAM-1 were remarkably higher in hyperlipidemic subjects compared to control. Correlation analyses demonstrated that Gla-Gas6 levels were inversely correlated with MCP-1 and ICAM-1 but positively with plasma VK in hyperlipidemic subjects but not in control. This suggests that boosting VK level might ameliorate the hyperlipidemia-associated inflammatory pathophysiology via augmenting Gla-Gas6. Further studies with high fat diet (HFD)-fed mice demonstrated that VK supplementation (1, 3, and 5 µg/kg BW, 8 weeks) dose-dependently reduced both hepatic and plasma levels of MCP-1 and ICAM-1 while elevating that of Gla-Gas6 but not total Gas6 in HFD-fed mice. Cell culture studies with gamma-glutamyl carboxylase (enzyme causes VK-dependent carboxylation of Gas6) knockdown hepatocytes and monocytes dissected the direct role of Gla-Gas6 in inhibiting high palmitic acid (0.75 mM)-induced inflammation via arresting MCP-1/ICAM-1 mediated hepatocyte-monocyte adhesion. The present study demonstrated an important role of Gla-Gas6 in facilitating the prophylactic effect of VK against hyperlipidemia associated inflammation.