VCAM-1 Protein Expression Research Articles

Sustainability of shear stress conditioning in endothelial colony-forming cells compared to human aortic endothelial cells to underline suitability for tissue-engineered vascular grafts

The endothelialization of cardiovascular implants is supposed to improve the long-term patency of these implants. In addition, in previous studies, it has been shown, that the conditioning of endothelial cells by dynamic cultivation leads to the expression of an anti-thrombogenic phenotype. For the creation of a tissue-engineered vascular graft (TEVG), these two strategies were combined to achieve optimal hemocompatibility. In a clinical setup, this would require the transfer of the already endothelialized construct from the conditioning bioreactor to the patient. Therefore, the reversibility of the dynamic conditioning of the endothelial cells with arterial-like high shear stress (20 dyn/cm2) was investigated to define the timeframe (tested in a range of up to 24 h) for the perseverance of dynamically induced phenotypical changes. Two types of endothelial cells were compared: endothelial colony-forming cells (ECFCs) and human aortic endothelial cells (HAECs). The results showed that ECFCs respond far more sensitively and rapidly to flow than HAECs. The resulting cell alignment and increased protein expression of KLF-2, Notch-4, Thrombomodulin, Tie2 and eNOS monomer was paralleled by increased eNOS and unaltered KLF-2 mRNA levels even under stopped-flow conditions. VCAM-1 mRNA and protein expression was downregulated under flow and did not recover under stopped flow. From these time kinetic results, we concluded, that the maximum time gap between the TEVG cultivated with autologous ECFCs in future reactor cultivations and the transfer to the potential TEVG recipient should be limited to ∼6 h.

Protective effect of rubber seed oil on human endothelial cells

ObjectiveThis study was conducted to characterize the antioxidant and anti-inflammatory properties of Rubber Seed Oil (RSO) against atherosclerosis (AS) through the study of the protective effects and mechanisms on human umbilical vein endothelial cells (HUVECs) injury induced by oxidized low-density lipoprotein (ox-LDL).MethodsHUVECs were treated with RSO, ox-LDL, RSO + ox-LDL, respectively, followed by cell activity testing, levels of IL-1β, IL-6, IL-10, TNF-α, ROS, NO, the mRNA expression of eNOS and protein expression of MCP-1, VCAM-1, eNOS, TLR4, NF-κB p65、p-NF-κB p65.ResultsCompared with the ox-LDL group, cell viability, NO level and the expression of eNOS mRNA significantly increased. and the levels of pro-inflammatory factors such as IL-1β, IL-6, TNF-α, IL-10, ROS were significantly decreased, which was accompanied by decreases in TLR4 mRNA, TLR4, MCP-1, VCAM-1 protein expression, as well as the ratio of NF-κB p-p65/p65 in the group treated with 250 μg/ml ox-LDL + 50 μg/ml RSO, 250 μg/ml ox-LDL + 100 μg/ml RSO, 250 μg/ml ox-LDL + 150 μg/ml RSO.ConclusionsRSO can reduce the expression of pro-inflammatory mediators, oxidative factors involved in injured vascular endothelial cells, exhibiting anti-inflammatory and antioxidant properties HUVECs exposed to ox-LDL. In addition, it may alleviate endothelial cell damage by inhibiting the TLR4/NF-κB signaling pathway.

Investigating the interplay between LIGHT and HIF in inflammatory fibroblast responses

Introduction: Intestinal fibroblast differentiation into an inflammatory phenotype likely plays a key role in Inflammatory Bowel Disease (IBD). However, the mechanisms that control this process are poorly understood. The cytokine TNFSF14 (LIGHT) drives inflammatory fibroblast responses in other diseases such as Eosinophilic esophagitis. Additionally, hypoxia, defined as low cellular or tissue levels of oxygen, is a feature of IBD progression. The use of hydroxylase inhibiting drugs which exhibit hypoxia mimetic conditions have been shown in murine models to have protective effects in IBD. Here we hypothesize the role of LIGHT to be of central importance in intestinal fibroblast differentiation and test how this inflammatory phenotype may be affected by hypoxia mimetic drugs and conversely what effect LIGHT may have on the hypoxic condition. Methods: Primary human colonic fibroblasts (18CO), the hepatic stellate cell line (LX2) and the epithelial cell line (T84) were treated with LIGHT in time course studies. 18COs were also treated with hydroxylase inhibitors Dimethyloxalylglycine (DMOG), JNJ42041935 and Roxadustat. Responses were analysed using qPCR, flowcytometry and Immunoblot-analysis. Results: LIGHT-treated 18COs upregulated CCL2 (7-fold ± increase, p=<0.001, n=5), CXCL10 (14-fold ± increase, p=<0.0001, n=3) and IL34 (30-fold ± increase, p=<0.05 n=5) at transcript level, and surface protein expression of ICAM1 (15% ± increase at 24 hours compared to vehicle, n=4) and VCAM1 (18% ± increase at 24 hours compared to vehicle, n=4) displaying distinct kinetics. This response was absent or to a much less significant degree in LX2s and T84s. DMOG decreased CCL2 (4-fold ± decrease compared to LIGHT treated samples, p=<0.001, n=5) and CXCL10 (11-fold ± decrease compared to LIGHT treated samples, p=<0.0001, n=3) gene expression in 18COs when subsequently treated with LIGHT for 8 hours but did not affect other targets tested such as CSF1, CXCL5, VCAM1 and ICAM1. Immunoblot analysis of 18COs showed a decreased HIF-1α stabilization when treated with hydroxylase inhibitors in the presence of LIGHT compared to hydroxylase inhibitors alone (3 different hydroxylase inhibiting drugs n=3-4, n=11 total). Preliminary data of HIF downstream activated genes VEGFA (14-fold ± increase, p=<0.05 n=3) and GLUT1 (6-fold ± increase, p=<0.05 n=3) show increased gene expression in 18COs when treated with DMOG with noticeable decreases when treated with a LIGHT DMOG combination. Conclusions: Our current studies show that LIGHT is a potential mediator of colonic inflammatory fibroblast differentiation and a possible inhibitor of HIF-1α stabilization. The effects of LIGHT are either significantly weaker or not displayed in colonic epithelial cells or hepatic stellate cells, indicating LIGHT-specific effects on intestinal stroma. Hydroxylase inhibitors show selective effects on the LIGHT driven response. Collectively our data reveals a potential LIGHT-hypoxia network that may be amenable for therapeutic interventions. SFI/IRC Pathway Fellows Award to Dr. Manresa, 21/PATH-S/9621. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Diosgenin protects against cationic bovine serum albumin-induced membranous glomerulonephritis by attenuating oxidative stress and renal inflammation via the NF-κB pathway

Context Membranous glomerulonephritis (MGN) is a leading cause of nephrotic syndrome in adults. Diosgenin (DG) has been reported to exert antioxidative and anti-inflammatory effects. Objective To investigate the renoprotective activity of DG in a cationic bovine serum albumin-induced rat model of MGN. Materials and methods Fourty male Sprague-Dawley rats were randomized into four groups. The MGN model was established and treated with a DG dose (10 mg/kg) and a positive control (TPCA1, 10 mg/kg), while normal control and MGN groups received distilled water by gavage for four consecutive weeks. At the end of the experiment, 24 h urinary protein, biochemical indices, oxidation and antioxidant levels, inflammatory parameters, histopathological examination, immunohistochemistry and immunoblotting were evaluated. Results DG significantly ameliorated kidney dysfunction by decreasing urinary protein (0.56-fold), serum creatinine (SCr) (0.78-fold), BUN (0.71-fold), TC (0.66-fold) and TG (0.73-fold) levels, and increasing ALB (1.44-fold). DG also reduced MDA (0.82-fold) and NO (0.83-fold) levels while increasing the activity of SOD (1.56-fold), CAT (1.25-fold), glutathione peroxidase (GPx) (1.55-fold) and GSH (1.81-fold). Furthermore, DG reduced Keap1 (0.76-fold) expression, Nrf2 nuclear translocation (0.79-fold), and induced NQO1 (1.25-fold) and HO-1 (1.46-fold) expression. Additionally, DG decreased IL-2 (0.55-fold), TNF-α (0.80-fold) and IL-6 (0.75-fold) levels, and reduced protein expression of NF-κB p65 (0.80-fold), IKKβ (0.93-fold), p-IKKβ (0.89-fold), ICAM-1 (0.88-fold), VCAM-1 (0.91-fold), MCP-1 (0.88-fold) and E-selectin (0.87-fold), and also inhibited the nuclear translocation of NF-κB p65 (0.64-fold). Discussion and conclusions The results suggest a potential therapeutic benefit of DG against MGN due to the inhibition of the NF-κB pathway, supporting the need for further clinical trials.

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

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

Modulation of the Cardiovascular Effects of Polycyclic Aromatic Hydrocarbons: Physical Exercise as a Protective Strategy.

Exposure to polycyclic aromatic hydrocarbons (PAHs) present in air pollution increases cardiovascular risk. On the contrary, physical exercise is a widely used therapeutic approach to mitigate cardiovascular risk, but its efficacy in an environment of air pollution, particularly with PAHs, remains unclear. This study investigates the effects of exercise on inflammation, endothelial dysfunction, and REDOX imbalance due to PAH exposure using a mouse model. Twenty male BALB/c mice were subjected to a mixture of PAHs (phenanthrene, fluoranthene, pyrene) in conjunction with aerobic exercise. The investigation evaluated serum levels of inflammatory cytokines, gene expression linked to inflammatory markers, endothelial dysfunction, and REDOX imbalance in aortic tissues. Furthermore, the study evaluated the expression of the ICAM-1 and VCAM-1 proteins. Exercise led to notable changes in serum inflammatory cytokines, as well as the modulation of genes associated with endothelial dysfunction and REDOX imbalance in aortic tissue. In turn, exercise produced a modulation in the protein expression of ICAM-1 and VCAM-1. The findings implicate the potential of exercise to counter PAH-induced damage, as demonstrated by changes in markers. In conclusion, exercise could mitigate the adverse effects related to exposure to PAHs present in air pollution, as evidenced by changes in inflammatory markers, endothelial dysfunction, and REDOX imbalance.

Inducible nitric oxide synthase activity mediates TNF-α-induced endothelial cell dysfunction.

Inducible nitric oxide synthase (iNOS) and vascular endothelial dysfunction have been implicated in the development and progression of atherosclerosis. This study aimed to elucidate the role of iNOS in vascular endothelial dysfunction. Ultrahigh performance liquid chromatography-quadrupole time-of-flight mass spectrometry combined with multivariate data analysis was used to characterize the metabolic changes in human umbilical vein endothelial cells (HUVECs) in response to different treatment conditions. In addition, molecular biology techniques were employed to explain the molecular mechanisms underlying the role of iNOS in vascular endothelial dysfunction. Tumor necrosis factor-α (TNF-α) enhances the expression of iNOS, TXNIP, and the level of reactive oxygen species (ROS) facilitates the entry of nuclear factor-κB (NF-κB) into the nucleus and promotes injury in HUVECs. iNOS deficiency reversed the TNF-α-mediated pathological changes in HUVECs. Moreover, TNF-α increased the expression of tumor necrosis factor receptor-2 (TNFR-2) and the levels of p-IκBα and IL-6 proteins and CD31, ICAM-1, and VCAM-1 protein expression, which was significantly reduced in HUVECs with iNOS deficiency. In addition, treating HUVECs in the absence or presence of TNF-α or iNOS, respectively, enabled the identification of putative endogenous biomarkers associated with endothelial dysfunction. These biomarkers were involved in critical metabolic pathways, including glycosylphosphatidylinositol-anchor biosynthesis, amino acid metabolism, sphingolipid metabolism, and fatty acid metabolism. iNOS deficiency during vascular endothelial dysfunction may affect the expression of TNFR-2, vascular adhesion factors, and the level of ROS via cellular metabolic changes, thereby attenuating vascular endothelial dysfunction.NEW & NOTEWORTHY Inducible nitric oxide synthase (iNOS) deficiency during vascular endothelial dysfunction may affect the expression of tumor necrosis factor receptor-2 and vascular adhesion factors via cellular metabolic changes, thereby attenuating vascular endothelial dysfunction.

Truncated oxidized phospholipids exacerbate endothelial dysfunction and lung injury caused by bacterial pathogens

Oxidized phospholipids (OxPLs) are present at basal levels in circulation of healthy individuals, but a substantial increase and changes in composition of OxPLs may rapidly occur during microbial infections, sepsis, and trauma. Specifically, truncated oxidized phospholipids (Tr-OxPLs) exhibit detrimental effects on pulmonary endothelium, yet their role on modulation of lung injury caused by bacterial pathogens remains to be elucidated. This study investigated the effects of Tr-OxPL species: KOdiA-PC, POV-PC, PON-PC, PAz-PC, PGPC, and Lyso-PC on endothelial permeability and inflammatory responses to gram-positive bacterial particles. Results showed that all six tested Tr-OxPLs augmented endothelial barrier disruption caused by heat-killed Staphylococcus aureus (HKSA) as determined by VE-cadherin immunostaining and monitoring transendothelial electrical resistance. In parallel, even moderate elevation of Tr-OxPLs augmented HKSA-induced activation of NF-κB, secretion of IL-6 and IL-8, and protein expression of ICAM-1 and VCAM-1. In the mouse model of acute lung injury caused by intranasal injection of HKSA, intravenous Tr-OxPLs administration augmented HKSA-induced increase in BAL protein content and cell counts, tissue expression of TNFα, KC, IL1β, and CCL2, and promoted vascular leak monitored by lung infiltration of Evans Blue. These results suggest that elevated Tr-OxPLs act as critical risk factor worsening bacterial pathogen-induced endothelial dysfunction and lung injury.

Liraglutide ameliorates oxidized LDL-induced endothelial dysfunction by GLP-1R-dependent downregulation of LOX-1-mediated oxidative stress and inflammation

ABSTRACTObjectiveTo investigate the effects of glucagon-like peptide 1 receptor (GLP-1R) agonist liraglutide on endothelial dysfunction in LDL receptor-deficient (LDLR-KO) mice and ox-LDL-challenged human umbilical vein endothelial cells (HUVECs) and its possible mechanism.MethodsLDLR-KO mice were randomly treated with normal saline, liraglutide, or liraglutide plus a GLP-1R antagonist exendin-9 for four weeks. In parallel, HUVECs were cultured with ox-LDL alone or combined with liraglutide, in the presence or absence of lectin-like ox-LDL receptor-1(LOX-1) overexpression or GLP-1R knockdown. Endothelial-dependent relaxation and LOX-1 protein expression of thoracic aorta, circulating levels of oxidative and inflammatory markers in mice, and cell survival, reactive oxygen species production, and expression of adhesion molecules and signal regulators in ox-LDL cultured endothelial cells were measured.Resultsliraglutide effectively enhanced acetylcholine-induced vasodilation, reduced LOX-1 expression in aortas, and decreased circulatory oxidative and inflammatory levels in LDLR-KO mice, which were abolished by cotreatment with exendin-9. HUVECs exposed to ox-LDL exhibited reduced cell viability, increased reactive oxygen species production and apoptosis, and elevated protein expression of ICAM-1, VCAM-1, LOX-1, NOX4, and NF-κB, which were markedly ameliorated by liraglutide treatment. The protective effects of liraglutide against ox-LDL-induced cell injury were abrogated in HUVECs overexpressing LOX-1 or silencing GLP-1R.ConclusionsLiraglutide improved oxidized LDL-induced endothelial dysfunction via GLP-1R-dependent downregulation of LOX-1-mediated oxidative stress and inflammation.

Multi-omic analysis of the cardiac cellulome defines a vascular contribution to cardiac diastolic dysfunction in obese female mice

Coronary microvascular dysfunction (CMD) is associated with cardiac dysfunction and predictive of cardiac mortality in obesity, especially in females. Clinical data further support that CMD associates with development of heart failure with preserved ejection fraction and that mineralocorticoid receptor (MR) antagonism may be more efficacious in obese female, versus male, HFpEF patients. Accordingly, we examined the impact of smooth muscle cell (SMC)-specific MR deletion on obesity-associated coronary and cardiac diastolic dysfunction in female mice. Obesity was induced in female mice via western diet (WD) feeding alongside littermates fed standard diet. Global MR blockade with spironolactone prevented coronary and cardiac dysfunction in obese females and specific deletion of SMC-MR was sufficient to prevent obesity-associated coronary and cardiac diastolic dysfunction. Cardiac gene expression profiling suggested reduced cardiac inflammation in WD-fed mice with SMC-MR deletion independent of blood pressure, aortic stiffening, and cardiac hypertrophy. Further mechanistic studies utilizing single-cell RNA sequencing of non-cardiomyocyte cell populations revealed novel impacts of SMC-MR deletion on the cardiac cellulome in obese mice. Specifically, WD feeding induced inflammatory gene signatures in non-myocyte populations including B/T cells, macrophages, and endothelium as well as increased coronary VCAM-1 protein expression, independent of cardiac fibrosis, that was prevented by SMC-MR deletion. Further, SMC-MR deletion induced a basal reduction in cardiac mast cells and prevented WD-induced cardiac pro-inflammatory chemokine expression and leukocyte recruitment. These data reveal a central role for SMC-MR signaling in obesity-associated coronary and cardiac dysfunction, thus supporting the emerging paradigm of a vascular origin of cardiac dysfunction in obesity.

Follicle-Stimulating Hormone Accelerates Atherosclerosis by Activating PI3K/Akt/NF-κB Pathway in Mice with Androgen Deprivation

Objective: Follicle-stimulating hormone (FSH) level changes may be another reason for increasing the risk of cardiovascular disease. In this study, we aimed to investigate the role of FSH in atherosclerosis and its underlying mechanism. Methods: ApoE^−/− mice were divided into 4 groups, namely, the sham group, bilaterally orchidectomized group, FSH group, and testosterone-only group. Blood lipid and hormone levels were tested, aorta Oil Red O staining; the levels of NF-κB, Akt, eNOS, and FSH receptors in the aorta were measured by Western blotting. Expression of VCAM-1 was detected via Western blotting and immunohistochemical staining. Human umbilical vein endothelial cells (HUVECs) were used to induce endothelial injury model by adding FSH, and the levels of NF-κB, Akt, eNOS, and FSHR were tested in HUVECs. Results: FSH treatment exacerbated atherosclerotic lesions in ApoE^−/− mice. Moreover, FSH could promote the expression of VCAM-1 protein in HUVECs, and this effect was possibly mediated by the activation of NF-κB, while NF-κB activation was further enhanced by the activation of the PI3K/Akt/eNOS pathway. FSH failed to activate Akt and NF-κB in the presence of the PI3K inhibitor LY294002 in HUVECs. Conclusion: FSH promoted the development of atherosclerosis by increasing VCAM-1 protein expression via activating PI3K/Akt/NF-κB pathway.

Histone Deacetylase 6 Inhibition Prevents Vaso-Occlusion and Pain in Sickle Cell Mice

Introduction: In sickle cell disease (SCD) mouse models, heme activates toll-like receptor 4 (TLR4) MyD88-dependent signaling and activates NOD-like receptor family pyrin domain containing (NLRP) 3 inflammasome assembly, leading to vascular endothelial activation and vaso-occlusion (VO). In SCD patients, cycles of hemolysis-driven VO contribute to acute and chronic pain/hyperalgesia. Several studies have evaluated non-selective histone deacetylase (HDAC) inhibitors for SCD; however, side-effects have limited clinical advancement. HDAC6 is an attractive target as it exhibits cytoplasmic AND nuclear activity and knockout mice lack hematologic consequences. In the cytoplasm, HDAC6 helps assemble NLRP3 inflammasomes and functions downstream of TLR4 to inhibit signaling. In SCD mouse models, TLR4 signaling is also critical for mechanical hyperalgesia. Intriguingly, in non-SCD hyperalgesia models, HDAC6 inhibition reduces TLR4-mediated mechanical hypersensitivity. We hypothesized that by preventing TLR4/NLRP3 activation in SCD mice, HDAC6 inhibition will prevent VO and pain. Methods: For in vitro analysis, TLR-4 signaling was stimulated in human umbilical vein endothelial cells (HUVEC) or mouse microglial cells (SIM-A9, ATTC) using 10-20 µM hemin or 10 ng/mL lipopolysaccharide (LPS) ± HDAC6 inhibitor BML-281 (2 µM, Enzo). To assess for pro-adhesive (VCAM-1, P-selectin, VWF) and pro-inflammatory (IL-6, NF-κB, IL-1) gene and protein expression, cell lysates or conditioned media were collected for analysis using qRT-PCR, immunoblotting, and enzyme-linked immunosorbent assay. Immunofluorescence (IF) for P-selectin and VWF was performed on fresh frozen organ sections or cells fixed with 4% paraformaldehyde. To assess for reactive oxygen species generation (ROS), the TMRE assay was used (Abcam). For dorsal skin fold chamber (DSFC) assessment of VO, HbSS Townes mice (n=7 per condition) were subcutaneously injected with either vehicle (DMSO) or BML-281 (1 mg/kg) 24h and 1h prior to DSFC implantation. After DSFC implantation, baseline venules were assessed. To initiate VO, panhematin (3.2 µmole/kg) was infused via tail vein, and at 1-4 hours post-infusion venules were reexamined for stasis. Mice were then sacrificed and organs stored for pathology assessment. For pain studies, HbSS Townes mice (n=6) were assessed for mechanical hyperalgesia for 4 days prior to injections. Only non-hyperalgesic mice were injected subcutaneously with either vehicle (DMSO, n=3) or BML-281 (1 mg/kg, n=3) 24h and 1h prior to panhematin infusion. To induce acute pain, panhematin (3.2 µmole/kg) was infused via tail vein, and 1h, 4h, 24h and 48h post-infusion mechanical hyperalgesia measurements were performed. Results: Compared to LPS or hemin treated ECs, addition of 2 µM BML-281 prevented upregulation of , VCAM-1, P-selectin and E-selection mRNA, this was inhibited by the addition of 2 µM BML-281. Likewise, compared to hemin-treated ECs, BML-281+hemin-treated ECs had reduced pro-adhesive VCAM-1 and E-selectin protein expression. In IF studies, ECs treated with BML-281+hemin had reduced VWF expression and P-selectin release. Hemin-treated HUVEC exhibit increased NLRP3-mediated ROS production, which BML-281 treatments was significantly decreased. In DSFC studies, compared to vehicle-treated mice, BML-281-treated SS Townes mice had significantly fewer static vessels after panhematin infusion (Fig 1). In pathology assessment, BML-281 treated mice, showed reduced lung VWF and P-selectin vessel deposition. To assess neuroinflammation, mouse microglial cells were treated with LPS. Compared to LPS alone, BML-281 treatment significantly reduced IL-6 secretion and NLRP3 mRNA upregulation. Importantly, in in vivo pain assessments, compared to vehicle-treated SS-Townes mice at 1 h and 4 h post-hemin infusion, BML-281-treated SS-Townes mice exhibited significantly less mechanical hyperalgesia. Conclusion: Our studies show that HDAC6 inhibition prevented hemin-mediated VO, potentially through reduction in hemin-mediated ROS and TLR4 driven pro-adhesive endothelial activation. Furthermore, HDAC6 inhibition prevented hemin-mediated pro-inflammatory microglial activation and reduced hemin-mediated acute pain stimulation. Collectively, these studies suggest that selective HDAC6 inhibition may be a strategy to reduce acute inflammation and pain in SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EFFECTS OF PALM OIL DERIVED TOCOTRIENOL RICH FRACTION AND VITAMIN E ISOMERS ON BIOMARKERS OF EARLY ATHEROGENESIS IN STIMULATED HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS

This study was conducted to investigate the effects of tocotrienol rich fraction (TRF), α-TOC, and pure TCT isomers (α-. γ- & δ-TCT) on inflammation, endothelial activation, nuclear factor kappa B (NFκB), endothelial nitric oxide synthase (eNOS) and monocyte binding activity (MBA) in vitro. Human umbilical vein endothelial cells (HUVECs) were incubated with various concentrations of α-TOC, pure TCT isomers and TRF (0.3-10 µM) together with lipopolysaccharides (LPS) for 16 h. Culture medium and cells were collected and measured for the protein and gene expression of IL-6, TNF-α, NFκB, ICAM-1, VCAM-1, e-selectin, and eNOS. Monocyte binding activity (MBA) was measured by Rose Bengal staining. Area under the curve (AUC) analysis revealed that TRF and pure TCT particularly γ- and δ- isomers, showed better inhibition of inflammation and endothelial activation, MBA and greater eNOS increment than α-TOC. These suggest that TRF and pure TCT isomers have potential as preventive anti-atherogenic agents by attenuating the release of early biomarkers of atherogenesis which is better than α-TOC in LPS-stimulated human endothelial cells.

Baiying qingmai formulation ameliorates thromboangiitis obliterans by inhibiting HMGB1/RAGE/NF-κB signaling pathways.

Baiying Qingmai Formulation (BF) is a classical clinical prescription used for decades to treat thromboangiitis obliterans (TAO). Although it effectively relieves pain and ischemic ulcers in patients with TAO, its anti-TAO mechanisms remain unclear. The chemical components of BF were analyzed using high-performance liquid chromatography and the potential targets of the compounds identified in BF were analyzed using molecular docking. Further, the signaling pathways and molecular mechanism of BF in treating TAO were studied using a rat model of TAO. Seven compounds (gallic acid, catechin, chlorogenic acid, caffeic acid, paeoniflorin, quercetin, and paeonol) were identified in BF, and molecular docking predicted their high affinities with HMGB1/RAGE/NF-κB proteins. In in vivo studies, BF not only inhibited the protein expression of HMGB1, RAGE, ICAM-1, and VCAM-1; mRNA levels of HMGB1 and RAGE; and the phosphorylation of NF-κB, ERK, Janus kinase (JNK) and p38 MAPK in the femoral artery, but also reduced the levels of inflammatory cytokines (IL-6, TNF-α, IL-1β, HMGB1) and stable metabolite (TXB2) of cytokine promoting thrombosis (TXA2) in the plasma. Moreover, BF stimulated the secretion of stable metabolite (6-keto-PGF1α) of cytokine inhibiting thrombosis (PGI2) in the plasma. BF inhibited the inflammatory response and thrombosis in the femoral artery, thus reducing the degree of vascular occlusion, which alleviated the symptoms in rats with TAO. Our findings suggest that BF ameliorates TAO by inhibiting the activation of the ERK, JNK, p38 MAPK and HMGB1/RAGE/NF-κB signaling pathways, thereby providing novel ideas for the treatment of TAO and essential information for the further development and utilization of BF as a promising drug to treat TAO.

Circulating extracellular histones exacerbate acute lung injury by augmenting pulmonary endothelial dysfunction via TLR4-dependent mechanism.

Extracellular histones released into the circulation following trauma, sepsis, and ARDS may act as potent damage-associated molecular pattern signals leading to multiple organ failure. Endothelial cell (EC) dysfunction caused by extracellular histones has been demonstrated in vitro and in vivo; however, precise mechanistic details of histone-induced EC dysfunction and exacerbation of ongoing inflammation remain poorly understood. This study investigated the role of extracellular histones in exacerbating preexisting endothelial dysfunction and acute lung injury. Histone subunits H3 and H4, but not H1, H2A, or H2B, induced permeability in human pulmonary EC. H3 and H4 at concentrations above 30 µg/mL caused EC inflammation reflected by activation of the NF-κB pathway, transcriptional activation, and release of cytokines and chemokines including IL-6 and IL-8, and increased mRNA and protein expression of EC adhesion molecules VCAM-1 and ICAM-1. Pharmacological inhibitors targeting Toll-like receptor TLR4 but not TLR2/6, blocked histone-induced EC dysfunction. H3 and H4 also strongly augmented EC permeability and inflammation caused by Gram-negative and Gram-positive bacterial particles, endotoxin, and TNFα. Heparin blocked histone-induced augmentation of EC inflammation caused by endotoxin and TNFα. Injection of histone in mouse models of lung injury caused by bacterial wall lipopolysaccharide (LPS) and heat-killed Staphylococcus aureus (HKSA) augmented ALI parameters: increased protein content, cell count, and inflammatory cytokine secretion in bronchoalveolar lavage fluid. Important clinical significance of these findings is in the demonstration that even a modest increase in extracellular histone levels can act as a severe exacerbating factor in conjunction with other EC barrier disruptive or proinflammatory agents.

GPR68 Inhibition with a Novel Group of Ogremorphin Inhibitors Upregulate Endothelial Barrier Function and Protect Against Bacterial Pathogens or Acidosis‐induced Inflammation in Lung Endothelium

BackgroundEndothelial dysfunction characterized by an increase in endothelial permeability and inflammatory responses are two major pathological hallmarks of various lung disorders including the current global pandemic COVID‐19. Thus, drugs targeting the preservation and restoration of endothelial function represent an attractive therapeutic target to treat endothelial dysfunction‐derived cardiopulmonary diseases. G protein‐coupled receptors (GPCRs), especially a sub‐family of proton‐sensing GPCRs including GPR4 and GPR68, have been recently suggested to play a role in the modulation of endothelial function. In this study, we investigated the barrier protective and anti‐inflammatory effects of two recently developed novel class of GPR68 inhibitors: ogremorphins OGM8345 and OGM‐1 in cultured human lung endothelial cells as well as in mice.MethodsEndothelial permeability was measured by monitoring transendothelial electrical resistance (TER) across human pulmonary arterial endothelial cells (HPAECs). Protein and mRNA expression levels of endothelial inflammation markers were analyzed by western blot and quantitative real time PCR, respectively. Acidosis was induced by switching cells to acidic pH (6.5) medium and luciferase‐based Tango assay was performed to evaluate GPR68 activation. C57BL/6 mice were exposed to lipopolysaccharide (LPS from Escherichia coli) or heat‐killed Staphylococcus aureus (HKSA), and vascular leak/inflammation was assessed by determining the extravasation of intravenously injected Evans blue tracer into lungs and total cells/protein count in bronchoalveolar lavage samples.ResultsBoth OGM8345 (1‐5 µM) and OGM‐1 (0.3‐1.5 µM) induced a robust dose‐dependent increase in basal EC barrier function that was evident by an 150‐200% increase in TER values. Both inhibitors also markedly attenuated LPS‐ and HKSA‐induced endothelial hyperpermeability. LPS or HKSA‐induced upregulation of inflammatory cytokines/chemokines genes TNF‐α, ICAM‐1, VCAM‐1, IL‐6, IL‐8, IL‐1β, and CXCL5 was significantly attenuated by OGMs as demonstrated by RT‐PCR analysis. Consistently, both OGMs suppressed LPS‐ and HKSA‐induced protein expression of phospho‐NFkB, VCAM‐1 and ICAM‐1. In contrast, pharmacologic inhibition of GPR4 by NE 52‐QQ57 failed to alleviate LPS or HKSA‐induced EC barrier dysfunction and inflammation. Importantly, LPS, HKSA or acidosis stimulation resulted in increased GPR68 mRNA expression as well as GPR68 activity that was inhibited by OGMs. Both OGMs attenuated vascular leak and lung inflammation caused by intratracheal injection of LPS or HKSA in C57BL/6 mice as illustrated by reduced Evans blue accumulation in the lungs and significant inhibition of inflammatory cells and protein content in bronchoalveolar lavage samples.ConclusionAltogether, these results establish an essential role of GPR68 in endothelial dysfunction and strongly suggest a therapeutic potential of GPR68‐selective inhibitors in improving endothelial dysfunction‐derived lung injuries.

Guanxinping ameliorates atherosclerosis via MAPK/NF-κB signaling pathway in ApoE-/- mice.

Atherosclerosis (AS), one of the leading causes of deaths and disabilities, is a kind of vascular disease of lipid disorders and chronic inflammation. Guanxinping (GXP) has been administrated in the treatment of AS for nearly 20years with satisfying clinical response. This study aimed to explore its underlying mechanisms of anti-atherosclerotic effect in AS. Male ApoE-/- mice were randomized into five groups and fed with either standard diet (control group, CON) or high-fat diet (HFD) for 12weeks. HFD mice were further divided randomly and either fed continually with HFD as a model group, or atorvastatin (ATO), or low-dose GXP (LGXP), or high-dose GXP (HGXP). After 12weeks, the body weight, serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c) were detected. Moreover, serum inflammation cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) concentrations were measured. The structure of aortic tissues was examined by hematoxylin-eosin staining. The mRNA expression of TNF-α, IL-6, and IL-1β were assessed by qPCR. The protein expressions of ICAM-1, VCAM-1, MCP-1, IL-6, IL-1β, p38MAPK, ERK1/2, JNK, IκB-α, and NF-κBp65 in the aorta were also detected. GXP treatment reduced serum TG, TC, and LDL-c levels in ApoE-/- mice. Moreover, GXP reduced lipid accumulation in the aorta of ApoE-/- mice, induced by HFD. Furthermore, GXP ameliorated the aorta morphological damage and reduced the serum TNF-α, IL-6, and IL-1β levels. GXP also attenuated the protein expression of ICAM-1, VCAM-1, MCP-1, IL-6, IL-1β, p38MAPK, ERK1/2, JNK, and NF-κBp65, whereas it increased the IκBα level in aortic tissues of ApoE-/- mice. Our results show that GXP could ameliorate atherosclerosis, which is mediated by inhibition of the MAPK/NF-κB signaling pathway in ApoE-/- mice. This study provides evidence that GXP might be a promising drug for the treatment of AS.

Effect of Auraptene on angiogenesis in Xenograft model of breast cancer.

Angiogenesis is the most important challenge in breast cancer treatment. Recently, scientists become interesting in rare natural products and intensive researches was performed to identify their pharmacological profile. Auraptene shows helpful effects such as cancer chemo-preventive, anti-inflammatory, anti-oxidant, immuno-modulatory. In this regard, we investigated the anti-angiogenesis effect of Auraptene in in-vitro and in-vivo model of breast cancer. In this study, 4T, MDA-MB-231 and HUVEC cell lines were used. The proliferation study was done by MTT assay. For tube formation assay, 250 matrigel, 1×104 HUVEC treated with Auraptene, 20ng/mL EGF, 20ng/mL bFGF and 20ng/mL VEGF were used. Gene expression of important gene related to angiogenesis in animal model of breast cancer was investigated by Real-time PCR. Protein expression of VCAM-1 and TNFR-1 gene related to angiogenesis in animal model of breast cancer was investigated by western-blot. Auraptene treatment led to reduction in cell viability of MDA-MB-231 in a concentration-dependent manner. Also, we observed change in the number of tubes or branches formed by cells incubated with 40 and 80μM Auraptene. Auraptene effect the gene expression of important gene related to angiogenesis (VEGF, VEGFR2, COX2, IFNɣ). Moreover, the western blot data exhibited that Auraptene effect the protein expression of VCAM-1 and TNFR-1. Overall, this study shows that Auraptene significantly suppressed angiogenesis via down-regulation of VEGF, VEGFR2, VCAM-1, TNFR-1, COX-2 and up-regulation of IFNγ.

Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation

NADPH oxidase 4 (NOX4) regulates endothelial inflammation by producing hydrogen peroxide (H2O2) and to a lesser extent O2•-. The ratio of NOX4-derived H2O2 and O2•- can be altered by coenzyme Q (CoQ) mimics. Therefore, we hypothesize that cytochrome b5 reductase 3 (CYB5R3), a CoQ reductase abundant in vascular endothelial cells, regulates inflammatory activation. To examine endothelial CYB5R3 in vivo, we created tamoxifen-inducible endothelium-specific Cyb5r3 knockout mice (R3 KO). Radiotelemetry measurements of systolic blood pressure showed systemic hypotension in lipopolysaccharides (LPS) challenged mice, which was exacerbated in R3 KO mice. Meanwhile, LPS treatment caused greater endothelial dysfunction in R3 KO mice, evaluated by acetylcholine-induced vasodilation in the isolated aorta, accompanied by elevated mRNA expression of vascular adhesion molecule 1 (Vcam-1). Similarly, in cultured human aortic endothelial cells (HAEC), LPS and tumor necrosis factor α (TNF-α) induced VCAM-1 protein expression was enhanced by Cyb5r3 siRNA, which was ablated by silencing the Nox4 gene simultaneously. Moreover, super-resolution confocal microscopy indicated mitochondrial co-localization of CYB5R3 and NOX4 in HAECs. APEX2-based electron microscopy and proximity biotinylation also demonstrated CYB5R3's localization on the mitochondrial outer membrane and its interaction with NOX4, which was further confirmed by the proximity ligation assay. Notably, Cyb5r3 knockdown HAECs showed less total H2O2 but more mitochondrial O2•-. Using inactive or non-membrane bound active CYB5R3, we found that CYB5R3 activity and membrane translocation are needed for optimal generation of H2O2 by NOX4. Lastly, cells lacking the CoQ synthesizing enzyme COQ6 showed decreased NOX4-derived H2O2, indicating a requirement for endogenous CoQ in NOX4 activity. In conclusion, CYB5R3 mitigates endothelial inflammatory activation by assisting in NOX4-dependent H2O2 generation via CoQ.

Vascular endothelial tissue factor contributes to trimethylamine N-oxide-enhanced arterial thrombosis.

Gut microbiota and their generated metabolites impact the host vascular phenotype. The metaorganismal metabolite trimethylamine N-oxide (TMAO) is both associated with adverse clinical thromboembolic events, and enhances platelet responsiveness in subjects. The impact of TMAO on vascular Tissue Factor (TF) in vivo is unknown. Here, we explore whether TMAO-enhanced thrombosis potential extends beyond TMAO effects on platelets, and is linked to TF. We also further explore the links between gut microbiota and vascular endothelial TF expression in vivo. In initial exploratory clinical studies, we observed that among sequential stable subjects (n = 2989) on anti-platelet therapy undergoing elective diagnostic cardiovascular evaluation at a single-site referral centre, TMAO levels were associated with an increased incident (3 years) risk for major adverse cardiovascular events (MACE) (myocardial infarction, stroke, or death) [4th quartile (Q4) vs. Q1 adjusted hazard ratio (HR) 95% confidence interval (95% CI), 1.73 (1.25-2.38)]. Similar results were observed within subjects on aspirin mono-therapy during follow-up [adjusted HR (95% CI) 1.75 (1.25-2.44), n = 2793]. Leveraging access to a second higher risk cohort with previously reported TMAO data and monitoring of anti-platelet medication use, we also observed a strong association between TMAO and incident (1 year) MACE risk in the multi-site Swiss Acute Coronary Syndromes Cohort, focusing on the subset (n = 1469) on chronic dual anti-platelet therapy during follow-up [adjusted HR (95% CI) 1.70 (1.08-2.69)]. These collective clinical data suggest that the thrombosis-associated effects of TMAO may be mediated by cells/factors that are not inhibited by anti-platelet therapy. To test this, we first observed in human microvascular endothelial cells that TMAO dose-dependently induced expression of TF and vascular cell adhesion molecule (VCAM)1. In mouse studies, we observed that TMAO-enhanced aortic TF and VCAM1 mRNA and protein expression, which upon immunolocalization studies, was shown to co-localize with vascular endothelial cells. Finally, in arterial injury mouse models, TMAO-dependent enhancement of in vivo TF expression and thrombogenicity were abrogated by either a TF-inhibitory antibody or a mechanism-based microbial choline TMA-lyase inhibitor (fluoromethylcholine). Endothelial TF contributes to TMAO-related arterial thrombosis potential, and can be specifically blocked by targeted non-lethal inhibition of gut microbial choline TMA-lyase.