ICAM-1 Expression Research Articles

Abstract P2152: Asperuloside Ameliorates Obesity-associated Endothelial Dysfunction Via Activating Nrf2/HO-1 Signaling Pathway

Objectives: Asperuloside (ASP) is the major bioactive components in Eucommia species but its effects on vascular health and the underlying mechanism remain unknown. In this study we aim to investigate the protective vascular effect of ASP and we hypothesize that ASP ameliorates obesity associated endothelial dysfunction. Methods: After induction of obesity in C57BL/6 male mice fed with high fat diet for twelve weeks, they were treated daily with ASP (50 mg/kg) via oral gavage for seven weeks in vivo study. C57BL/6 mice aortae were used for ex vivo study and endothelial cells were used for in vitro study. Vascular function was determined by wire myograph. DHE and MitoSOX staining were used to detect ROS levels. QPCR, aortic en-face immunofluorescence staining and Western blotting were used to analyze mRNA and protein expressions. Small interfering (si)RNA of Nrf2 and HO-1, inhibitors of Nrf2 (ML385) and HO-1 (OB-24) were used to elucidate the mechanistic pathway of ASP ’s action. Findings: ASP significantly improved endothelial-dependent relaxations, suppressed oxidative stress, reduced VCAM-1 (~2 fold) and ICAM-1 (~2 fold) expression, increased both Nrf2 (~1.5 fold) and HO-1 (~1.5 fold) expression in obese mice aortae, IL-1β-treated aortae ex vivo and endothelial cells. However, ASP failed to prevent endothelial dysfunction in HO-1 inhibited aortae and HO-1-siRNA transfected endothelial cells, indicating that ASP improved endothelial function through upregulating HO-1. Nrf2 knockdown attenuated HO-1 upregulation by ASP, showing that ASP regulated Nrf2/HO-1 pathway. ASP increased nuclear Nrf2 and dual luciferase reporter assay illustrated that ASP increased ARE binding activity. Moreover, inhibition of Nrf2/ARE binding by ML385 blocked vascular protective effects and HO-1 upregulation by ASP, showing that ASP activated Nrf2/HO-1 signaling by stimulating Nrf2/ARE binding. Conclusions: This study for the first time shows that ASP rescues endothelial dysfunction and suppresses vascular oxidative stress associated with obesity and IL-1β via activating Nrf2/HO-1 pathway. These findings explore the pharmacological effects of ASP and contribute to the development of a novel treatment for endothelial dysfunction in cardiovascular diseases.

Abstract P3064: Cytotoxic T-cells Drive Doxorubicin Induced Cardiac Fibrosis And Systolic Dysfunction

Background: Doxorubicin (DR), the most commonly prescribed chemotherapy, results in dose-dependent cardiotoxicity and heart failure (HF), and patients respond less frequently to standard HF treatments. DR causes oxidative stress and myocyte death, both potent inducers of a T-cell immune response and cardiac inflammation in other causes of HF. However, mechanistic depth on the immune response elicited by DR is limited. We hypothesized that an aberrant T-cell immune response secondary to cardiac damage by DR contributes to DR-induced HF and cardiac remodeling. Methods and Results: We treated wild type (WT) and T-cell deficient ( Tcra -/- ) mice with 5.0 mg/kg DR or PBS weekly by intraperitoneal injection for 4 or 8 weeks. We found at both time points Tcra -/- mice had preserved ejection fraction (EF) by echocardiography and decreased cardiac fibrosis by picrosirius red staining, compared to WT. At 8 weeks, Tcra -/- mice had fewer TUNEL + cardiac cells than WT mice. DR treated mice had increased frequency of activated effector CD8 + CD62L lo CD44 hi cells in the cardiac draining lymph nodes, increased circulating CD8 + T-cells, and cardiac CD8 + T-cell infiltration at both time points. Histologic analysis demonstrated localization of CD8 + T-cells adjacent to cardiac fibroblasts (CFB) in the hearts of DR treated mice. Targeted antibody depletion of CD8 + but not CD4 + cells prior to 4 week DR treatment resulted in improved EF and decreased cardiac fibrosis. Moreover, cardiac CD8 + T-cells expressed higher IFNγ in vivo in DR compared to PBS treated mice, and DR hearts had increased gene expression of the IFNγ inducible chemokines Cxcl9 and Cxcl10 . Ex vivo DR (0.1 μg/mL) treatment of CD8 + T-cells during TCR activation with αCD3/CD28 increased IFNγ expression at the protein and transcript level. The same dose of DR also increased surface expression of ICAM-1 on CFB, and CD8 + T-cells adhered to DR treated CFB in vitro in greater numbers than untreated CFB, and induced more αSMA expression. Lastly, in patients treated with DR, plasma levels of CXCL9 and CXCL10 correlated with decreases in EF after 3 months of treatment. Conclusion: These data outline a novel CD8 T-cell-driven inflammatory mechanism in DR cardiotoxicity, driven by IFNγ-inducible chemokines in animal models and patients.

Abstract P3107: Sexual Dimorphism In Diastolic Dysfunction And Cardiac Small Vessel Disease In Heart Failure With Preserved Ejection Fraction Mouse Models

Introduction: Coronary microvascular disease has been proposed to be responsible for heart failure with preserved ejection fraction (HFpEF) about 10 years ago. However, to date the role and phenotype of the coronary microvasculature has still been poorly investigated in animal models of HFpEF and sex differences are not always considered. Objective: To characterize the phenotype of the coronary microvasculature in male and female mice with diastolic dysfunction in two HFpEF mouse models. Method: We assessed cardiac function and characterized the coronary microvasculature in mice fed with a high fat diet (HFD) + L-NAME regimen and in Leptin receptor deficient ( Lepr db/db ) mice. Notably, our study was done in males, females and ovariectomized (OVX) female mice in order to search for possible sexual dimorphisms. Results: Upon a HFD + L-NAME regimen, both males and OVX females but not non OVX females develop diastolic dysfunction attested by an increased end diastolic pressure. In Lepr db/db mice, both Male and non OVX female mice develop diastolic dysfunction. Notably female mice have reduced estradiol and progesterone level mice mimicking ovariectomy. We found that both OVX and non OVX females but not males display increased endothelial activation attested by increased ICAM1 expression, endothelium leakage attested by increased Fibrinogen and IgG extravasation and decreased arteriole diameter suggesting vasoconstriction. The same results were found in Lepr db/db mice. Conclusion: Diastolic dysfunction is not always associated with cardiac small vessel disease since Lepr db/db males and C57BL/6J males fed with a high fat diet (HFD) + L-NAME regimen develop diastolic dysfunction in the absence of endothelial dysfunction. Also endothelial dysfunction may not be sufficient to induce diastolic dysfunction since non OVX female mice fed with a high fat diet (HFD) + L-NAME regimen display endothelial dysfunction while they do not develop diastolic dysfunction.

Abstract P2192: Growth Hormone-releasing Hormone Receptor Antagonist Attenuates Cardiopulmonary Injury Induced By A Bsl2 Rvsv-sars-cov-2-s Virus Mimic Of Sars-cov-2 Infectivity

Background: COVID-19 causes severe pulmonary injury that can lead to acute respiratory distress syndrome. Growth hormone-releasing hormone receptor (GHRH-R) and its splice variant are expressed in murine and human lung and heart. GHRH-R antagonist, MIA-602, has been shown to modulate cellular immune responses to bleomycin lung injury and decrease inflammation in models of sarcoid granuloma. Using the BSL2-friendly rVSV-SARS-CoV-2-S of K18 hACE2tg mice to mimic native SARS-CoV-2 infection, we tested our hypothesis that MIA-602 attenuates cardiopulmonary injury in this COVID-19 model. Methods: Male and female K18 hACE2tg mice were inoculated with SARS-CoV-2 Washington (WA-1) native strain, recombinant VSV-SARS-CoV-2-Spike virus (rVSV-SARS-CoV-2-S), or PBS and lung viral load, weight loss and histopathology compared between groups (N=5-8). K18 h ACE2 tg mice infected with rVSV-SARS-CoV-2-S were subject to daily subcutaneous injections of 10 μg MIA-602 or vehicle starting at 24h post-infection. Pulmonary function was measured via whole-body plethysmography on day 0, day 3, and day 5 (n=7). Five days after viral infection mice were sacrificed; and blood and tissues collected for histopathological analyses, H&E staining and ICAM-1 immunohistochemistry. T-test or One-way ANOVA-test was used for statistical analysis. Results: Native SARS-CoV-2 and rVSV-SARS-CoV-2-S presented with similar patterns of weight loss, infectivity (~60%) and histopathologic changes. Daily treatment with MIA-602 ameliorated weight loss, reduced lung perivascular inflammation and pneumonia, and decreased lung/heart ICAM-1 expression compared to vehicle. MIA-602 rescued respiratory rate, increased expiratory parameters (Te, PEF, EEP) and mitigated dysregulated measures of airway obstruction (Penh and Rpef) compared to vehicle. Conclusions: rVSV-SARS-CoV-2-S is an accurate and safe alternative to native SARS-CoV-2 for preclinical studies. Daily treatment with the synthetic peptide GHRH-R antagonist MIA-602 attenuates pulmonary dysfunction and heart inflammation in this new preclinical mouse model of COVID-19 pneumonia. We hypothesize that the molecular mechanism involves anti-inflammatory actions of MIA-602.

G/ β- TCP composite scaffold material promotes osteogenic differentiation of bone marrow mesenchymal stem cells.

To explore self-made graphene/β Graphene (G)/β- tricalcium phosphate, G/β- The effect of TCP composite scaffold material on osteogenic differentiation of BMSC. Preparation of G/β- TCP composite material was used to investigate the effect of composite material on bone marrow mesenchymal stem cell ossification/β- TCP material was used to treat primary BMSCs of rats. Cell morphology changes were observed under scanning electron microscopy, cell cycle and proliferation were detected by flow cytometry, and gene expression of chondrogenic genes Fibronectin, collagen I, collagen II, ICAM, and VCAM was detected by q-PCR. In addition, using osteogenic induction medium and G/β- TCP composite materials were co treated with BMSCs, and ALP and alizarin red staining were used to observe the effect of the materials on osteogenic differentiation. q-PCR was used to detect the gene expression of osteogenic related genes Runx2, OCN, and OPN. G/ β- After the TCP composite was co cultured with BMSC, the proportion of G0/G1 phase of BMSC cells was significantly increased, the cell proliferation ability was enhanced, and the gene expression of fibronectin, collagen I, collagen II, ICAM, and VCAM were significantly increased. The ALP staining results indicate that BMSC in G/β- After treatment with TCP composite material, significant enhancement of osteogenic ability was observed at 7,14 and 21 days. In addition, BMSC in G/β- A significant increase in calcium deposition was observed at 7,14 and 21 days after treatment with TCP composite materials. The effect of different time points on the expression of osteogenic related genes varies. At 7 and 14 days, the expression of RUNX2 was significantly reduced compared to the control, but significantly increased at 21 days; OCN significantly increased on the 21st day; OPN significantly increased at 14 days. G/β- TCP materials significantly promote the osteogenic differentiation of BMSCs.

Bradykinin B1 Receptor Affects Tumor-Associated Macrophage Activity and Glioblastoma Progression.

Bradykinin is a small active peptide and is considered an inflammatory mediator in several pathological conditions. Bradykinin exerts its effects by coupling to its receptors, including bradykinin B1 (B1R) and bradykinin B2. B1R has been implicated in the development of various cancers. Our previous study reported that B1R promoted glioblastoma (GBM) development by supporting the migration and invasion of GBM cells. However, the mechanisms underlying the effects of B1R on tumor-associated macrophages (TAMs) and GBM progression remain unknown. Accordingly, to explore the regulatory effects of B1R overexpression (OE) in GBM on tumor-associated immune cells and tumor progression, we constructed a B1R wild-type plasmid and developed a B1R OE model. The results reveal that B1R OE in GBM promoted the expression of ICAM-1 and VCAM-1-cell adhesion molecules-in GBM. Moreover, B1R OE enhanced GBM cell migration ability and monocyte attachment. B1R also regulated the production of the protumorigenic cytokines and chemokines IL-6, IL-8, CXCL11, and CCL5 in GBM, which contributed to tumor progression. We additionally noted that B1R OE in GBM increased the expression of CD68 in TAMs. Furthermore, B1R OE reduced the level of reactive oxygen species in GBM cells by upregulating heme oxygenase-1, an endogenous antioxidant protein, thereby protecting GBM cells from oxidative stress. Notably, B1R OE upregulated the expression of programmed death-ligand 1 in both GBM cells and macrophages, thus providing resistance against T-cell response. B1R OE in GBM also promoted tumor growth and reduced survival rates in an intracranial xenograft mouse model. These results indicate that B1R expression in GBM promotes TAM activity and modulates GBM progression. Therefore, B1R could be an effective target for therapeutic methods in GBM.

GM-CSF distinctly impacts human monocytes and macrophages via ERK1/2-dependent pathways

Human monocytes and macrophages are two major myeloid cell subsets with similar and distinct functions in tissue homeostasis and immune responses. GM-CSF plays a fundamental role in myeloid cell differentiation and activation. Hence, we compared the effects of GM-CSF on the expression of several immune mediators by human monocytes and monocyte-derived macrophages obtained from healthy donors. We report that GM-CSF similarly elevated the expression of CD80 and ICAM-1 and reduced HLA-DR levels on both myeloid cell subsets. However, GM-CSF increased the percentage of macrophages expressing surface IL-15 but reduced the proportion of monocytes carrying surface IL-15. Moreover, GM-CSF significantly increased the secretion of IL-4, IL-6, TNF, CXCL10, and IL-27 by macrophages while reducing the secretion of IL-4 and CXCL10 by monocytes. We show that GM-CSF triggered ERK1/2, STAT3, STAT5, and SAPK/JNK pathways in both myeloid subsets. Using a pharmacological inhibitor (U0126) preventing ERK phosphorylation, we demonstrated that this pathway was involved in both the GM-CSF-induced increase and decrease of the percentage of IL-15+ macrophages and monocytes, respectively. Moreover, ERK1/2 contributed to GM-CSF-triggered secretion of IL-4, IL-6, TNF, IL-27 and CXCL10 by macrophages. However, the ERK1/2 pathway exhibited different roles in monocytes and macrophages for the GM-CSF-mediated impact on surface makers (CD80, HLA-DR, and ICAM-1). Our data demonstrate that GM-CSF stimulation induces differential responses by human monocytes and monocyte-derived macrophages and that some but not all of these effects are ERK-dependent.

ICAM2 initiates trans-blood-CSF barrier migration and stemness properties in leptomeningeal metastasis of triple-negative breast cancer

Leptomeningeal metastasis (LM) occurs when tumor cells spread to the leptomeningeal space surrounding the brain and the spinal cord, thereby causing poor clinical outcomes. The triple-negative breast cancer (TNBC) has been associated with symptoms of LM and mechanism remained unclear. Through proteomic analysis, we identified high expression of ICAM2 in leptomeningeal metastatic TNBC cells, which promoted the colonization of the spinal cord and resulted in poor survival in vivo. Two-way demonstration indicated that high levels of ICAM2 promoted blood–cerebrospinal fluid barrier (BCB) adhesion, trans-BCB migration, and stemness abilities and determined the specificity of LM in vivo. Furthermore, pull-down and antibody neutralizing assay revealed that ICAM2 determined the specificity of LM through interactions with ICAM1 in the choroid plexus epithelial cells. Therefore, neutralizing ICAM2 can attenuate the progression of LM and prolong survival in vivo. The results suggested that targeting ICAM2 is a potential therapeutic strategy for LM in TNBC.

Astragaloside reduces toxic effect of periodontal ligament fibroblasts induced by lipopolysaccharide

Periodontitis is a non-specific and chronic disease which is highly prevalent, resulting in inflammation and destruction of periodontal tissues. This study aims to explore the effect and mechanism of astragaloside on periodontitis. We used CCK-8, Western Blot, qPCR and flow cytometry to analyze cell viability, related protein and mRNA expression, and cell apoptosis. We found that AST could promote cell proliferation and reduce apoptosis induced by LPS. Besides, AST could alleviate the increased expression of COX-2 and ICAM-1 induced by LPS. MiR-26b-3P specifically targeted the 3′ UTR of ICAM-1. These results indicate that AST reduces toxic effect of human periodontal ligament cells through regulating miR-26b-3P/ICAM-1, thus highlighting its protective role in periodontitis.

THE ROLE OF ICAM1 IN TUMOR ASSOCIATED MACROPHAGES IN GLIOBLASTOMA TUMORIGENESIS

Abstract Glioblastoma (GBM) is a fatal brain cancer in adults with ineffective treatment methods. Cell adhesion molecules (CAMs) are proteins that are expressed on the surface of cells and enable them to interact with one another and the surrounding microenvironment. Intracellular adhesion molecule 1 (ICAM-1) is a cell adhesion molecule expressed by various cell types. Preliminary data showed that ICAM-1 is associated with poorer overall and progression free survival in patients and ICAM-1 expression levels increase in recurrent tumors. TAMs enhance GBM tumor growth. The aim of this study was to determine if ICAM-1 expression on TAMs contributes to GBM tumorigenesis, specifically within the hypoxic tumor microenvironment. We hypothesized that ICAM1 facilitates TAM migration, proliferation, cell adhesion and phagocytosis, thus enhancing tumor growth. We found that upon incubation of human and mouse primary macrophages in hypoxia, ICAM-1 expression levels increased and were further exasperated upon culturing with tumor cell-conditioned medium. The migration, cell adhesion and phagocytosis of ICAM-1 deficient macrophages was lower than wild type macrophages, and effects were further exasperated upon culturing with tumor conditioned media and incubation in hypoxia. Intracranial injection of tumor cells into ICAM-1 deficient and wild type mice revealed that ICAM1 deficient mice survived longer with lower overall tumor volume than wild type mice, with survival and volume differences being rescued upon ICAM1 bone marrow reconstitution. In conclusion, the expression of ICAM1 in TAMs likely promotes GBM tumorigenesis and hypoxia further enhances the expression of ICAM-1 in macrophages which is associated with more aggressive GBM.

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.

Blood myeloid cells differentiate to lung resident cells and respond to pathogen stimuli in a 3D human tissue-engineered lung model.

Introduction: Respiratory infections remain a leading global health concern. Models that recapitulate the cellular complexity of the lower airway of humans will provide important information about how the immune response reflects the interactions between diverse cell types during infection. We developed a 3D human tissue-engineered lung model (3D-HTLM) composed of primary human pulmonary epithelial and endothelial cells with added blood myeloid cells that allows assessment of the innate immune response to respiratory infection. Methods: The 3D-HTLM consists of small airway epithelial cells grown at air-liquid interface layered on fibroblasts within a collagen matrix atop a permeable membrane with pulmonary microvascular endothelial cells layered underneath. After the epithelial and endothelial layers had reached confluency, an enriched blood monocyte population, containing mostly CD14+ monocytes (Mo) with minor subsets of CD1c+ classical dendritic cells (cDC2s), monocyte-derived dendritic cells (Mo-DCs), and CD16+ non-classical monocytes, was added to the endothelial side of the model. Results: Immunofluorescence imaging showed the myeloid cells migrate through and reside within each layer of the model. The myeloid cell subsets adapted to the lung environment in the 3D-HTLM, with increased proportions of the recovered cells expressing lung tissue resident markers CD206, CD169, and CD163 compared with blood myeloid cells, including a population with features of alveolar macrophages. Myeloid subsets recovered from the 3D-HTLM displayed increased expression of HLA-DR and the co-stimulatory markers CD86, CD40, and PDL1. Upon stimulation of the 3D-HTLM with the toll-like receptor 4 (TLR4) agonist bacterial lipopolysaccharide (LPS), the CD31+ endothelial cells increased expression of ICAM-1 and the production of IL-10 and TNFα was dependent on the presence of myeloid cells. Challenge with respiratory syncytial virus (RSV) led to increased expression of macrophage activation and antiviral pathway genes by cells in the 3D-HTLM. Discussion: The 3D-HTLM provides a lower airway environment that promotes differentiation of blood myeloid cells into lung tissue resident cells and enables the study of respiratory infection in a physiological cellular context.

Therapeutic potential of elema-1,3,7(11),8-tetraen-8,12-lactam from Curcuma wenyujin on diabetic retinopathy via anti-inflammatory and anti-angiogenic pathways

Ethnopharmacological relevanceIn traditional Chinese medicine, the causes of diabetic retinopathy (DR) are blood stasis and heat. Curcuma wenyujin Y. H. Chen & C. Ling and its extracts have the effects of promoting blood circulation to remove blood stasis, clearing the heart, and cooling the blood, and have been used in the treatment of DR. Elema-1,3,7 (11),8-tetraen-8,12-lactam (Ele), an N-containing sesquiterpene isolated from this plant. However, the anti-inflammatory and anti-angiogenic effects of Ele and its therapeutic potential in DR are still unknown. Aim of the studyTo evaluate the anti-inflammatory and anti-angiogenic effects of Ele and its therapeutic potential in DR. Materials and methodsIn vitro, anti-inflammatory and anti-angiogenic effects were assessed using TNF-α or VEGF-stimulated HUVECs. Protein expression was analyzed using Western blotting. ICAM-1 and TNF-α mRNA expressions were analyzed using real-time quantitative RT-PCR. The therapeutic potential in DR was assessed using both animal models of STZ-induced diabetes and oxygen-induced retinopathy. The retinal vascular permeability was measured using Evans blue, and the quantitation of retinal leukostasis using FITC-coupled Con A. The retinal neovascular tufts were analyzed using fluorescein angiography and counting pre-retinal vascular lumens. ResultsEle inhibited NF-κB pathway, and ICAM-1, TNF-α mRNA expression in TNF-α- stimulated HUVECs. It also inhibits the multistep process of angiogenesis by inhibiting the phosphorylation of VEGFR2 and its downstream signaling kinases Src, Erk1/2, Akt, and mTOR in VEGF-stimulated HUVECs. Intravitreal injection of Ele can significantly reduce retinal microvascular leakage, leukostasis, and expression of ICAM-1, TNF-α in diabetic rats and inhibits oxygen-induced retinal neovascularization and VEGFR2 phosphorylation in OIR mice. ConclusionsEle has anti-inflammatory and anti-angiogenic effects through inhibiting NF-κB and VEGFR2 signaling pathways, and it may be a potential drug candidate for DR.

Glycosaminoglycans and fucoidan have a protective effect on experimental glomerulonephritis.

Background: The glomerular endothelial glycocalyx is degraded during inflammation. The glycocalyx plays a pivotal role in endothelial function and is involved in many processes including binding of chemokines and cytokines, leukocyte trafficking, and preventing proteinuria. HS-based therapeutics are a promising novel class of anti-inflammatory drugs to restore a compromised endothelial glycocalyx under inflammatory conditions. Recently, we demonstrated that treatment with HS extracted from unstimulated glomerular endothelial glycocalyx (unstimulated HSglx) reduced albuminuria during anti-GBM induced glomerulonephritis. Since endothelial HS domains are distinct in unstimulated versus inflammatory conditions, we hypothesized that 1) unstimulated HSglx, 2) LPS-stimulated HSglx, 3) the HS-mimetic fucoidan and 4) the glycosaminoglycan preparation sulodexide, which is a mixture of low molecular weight heparin and dermatan sulfate, might have different beneficial effects in experimental glomerulonephritis. Methods: The effect of unstimulated HSglx, LPS HSglx, Laminaria japonica fucoidan, or sulodexide on experimental glomerulonephritis was tested in LPS-induced glomerulonephritis in mice. Analyses included urinary albumin creatinine measurement, cytokine expression in plasma and renal cortex, and renal influx of immune cells determined by flow cytometry and immunofluorescence staining. Furthermore, the observed in vivo effects were evaluated in cultured glomerular endothelial cells and peripheral blood mononuclear cells by measuring cytokine and ICAM-1 expression levels. The ability of the compounds to inhibit heparanase activity was assessed in a heparanase activity assay. Results: Treatment of mice with LPS HSglx or sulodexide near-significantly attenuated LPS-induced proteinuria. All treatments reduced plasma MCP-1 levels, whereas only fucoidan reduced IL-6 and IL-10 plasma levels. Moreover, all treatments reversed cortical ICAM-1 mRNA expression and both fucoidan and sulodexide reversed cortical IL-6 and nephrin mRNA expression. Sulodexide decreased renal influx of CD45+ immune cells whereas renal influx of macrophages and granulocytes remained unaltered for all treatments. Although all compounds inhibited HPSE activity, fucoidan and sulodexide were the most potent inhibitors. Notably, fucoidan and sulodexide decreased LPS-induced mRNA expression of ICAM-1 and IL-6 by cultured glomerular endothelial cells. Conclusion: Our data show a potentially protective effect of glycosaminoglycans and fucoidan in experimental glomerulonephritis. Future research should be aimed at the further identification of defined HS structures that have therapeutic potential in the treatment of glomerular diseases.

The Polyanionic Drug Suramin Neutralizes Histones and Prevents Endotheliopathy.

Drugs are needed to protect against the neutrophil-derived histones responsible for endothelial injury in acute inflammatory conditions such as trauma and sepsis. Heparin and other polyanions can neutralize histones but challenges with dosing or side effects such as bleeding limit clinical application. In this study, we demonstrate that suramin, a widely available polyanionic drug, completely neutralizes the toxic effects of individual histones, but not citrullinated histones from neutrophil extracellular traps. The sulfate groups on suramin form stable electrostatic interactions with hydrogen bonds in the histone octamer with a dissociation constant of 250 nM. In cultured endothelial cells (Ea.Hy926), histone-induced thrombin generation was significantly decreased by suramin. In isolated murine blood vessels, suramin abolished aberrant endothelial cell calcium signals and rescued impaired endothelial-dependent vasodilation caused by histones. Suramin significantly decreased pulmonary endothelial cell ICAM-1 expression and neutrophil recruitment caused by infusion of sublethal doses of histones invivo. Suramin also prevented histone-induced lung endothelial cell cytotoxicity invitro and lung edema, intra-alveolar hemorrhage, and mortality in mice receiving a lethal dose of histones. Protection of vascular endothelial function from histone-induced damage is a novel mechanism of action for suramin with therapeutic implications for conditions characterized by elevated histone levels.

The Nuclear Localization Signal of NF-κB p50 Enters the Cells via Syndecan-Mediated Endocytosis and Inhibits NF-κB Activity

It is well established that cationic peptides can enter cells following attachment to polyanionic membrane components. We report that the basic nuclear localization signal (NLS) of the NF-κB p50 subunit is internalized via lipid raft-dependent endocytosis mediated by heparan sulfate proteoglycans and exerts significant NF-κB inhibitory activities both in vitro and in vivo. In vitro uptake experiments revealed that the p50 NLS peptide (CYVQRKRQKLMP) enters the cytoplasm and accumulates in the nucleus at 37 °C. Depleting cellular ATP pools or decreasing temperature to 4 °C abolished peptide internalization, confirming the active, energy-dependent endocytic uptake. Co-incubation with heparan sulfate or replacing the peptide’s basic residues with glycines markedly reduced the intracellular entry of the p50 NLS, referring to the role of polyanionic cell-surface proteoglycans in internalization. Furthermore, treatment with methyl-β-cyclodextrin greatly inhibited the peptide’s membrane translocation. Overexpression of the isoforms of the syndecan family of transmembrane proteoglycans, especially syndecan-4, increased the cellular internalization of the NLS, suggesting syndecans’ involvement in the peptide’s cellular uptake. In vitro, p50 NLS reduced NF-κB activity in TNF-α-induced L929 fibroblasts and LPS-stimulated RAW 264.7 macrophages. TNF-α-induced ICAM-1 expression of HMEC-1 human endothelial cells could also be inhibited by the peptide. Fifteen minutes after its intraperitoneal injection, the peptide rapidly entered the cells of the pancreas, an organ with marked syndecan-4 expression. In an acute pancreatitis model, an inflammatory disorder triggered by the activation of stress-responsive transcription factors like NF-κB, administration of the p50 NLS peptide reduced the severity of pancreatic inflammation by blocking NF-κB transcription activity and ameliorating the examined laboratory and histological markers of pancreatitis.

Integration of network pharmacology and proteomics to elucidate the mechanism and targets of traditional Chinese medicine Biyuan Tongqiao granule against allergic rhinitis in an ovalbumin-induced mice model

Ethnopharmacological relevanceBiyuan Tongqiao granule (BYTQ) is a traditional Chinese medicine that has been used in China to clinically treat patients with allergic rhinitis (AR), yet its underlying mechanism and targets remains unclear. Aim of the studyThe study aimed to investigate the potential mechanism of BYTQ against AR using the ovalbumin (OVA) -induced AR mice model. Integrating network pharmacology and proteomics to investigate possible targets of BYTQ for AR. Materials and methodsThe compounds in BYTQ were analyzed using UHPLC-ESI-QE-Orbitrap-MS. The OVA/Al(OH)3 were used to induce the AR mice model. The nasal symptoms, histopathology, immune subsets, inflammatory factors, and differentially expressed proteins were examined. Proteomics analysis elucidated the potential mechanisms of BYTQ to improve AR, which was further validated by Western blot (WB) assay. The compounds and potential targets of BYTQ were systematically elucidated by integrating network pharmacology and proteomics analysis to explore the mechanism. The binding affinity between key potential targets and corresponding compounds was then validated using molecular docking. Molecular docking results were verified by a western blotting and cellular thermal shift assay (CETSA). ResultsA total of 58 compounds were identified from BYTQ. BYTQ significantly suppressed AR symptoms by inhibiting the release of OVA-specific immunoglobulin E (IgE) and histamine, improving the pathological injury of nasal mucosal tissue, and regulating the proportions of lymphocytes to maintain immune balance. Proteomics analysis showed that the cell adhesion factors and focal adhesion pathway might be potential mechanism of BYTQ against AR. The levels of E-selectin, vascular endothelial cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) proteins in the nasal mucosal tissue were significantly downregulated in the BYTQ-H group compared to the AR group. Integrating network pharmacology and proteomics analysis identified that SRC, PIK3R1, HSP90AA1, GRB2, AKT1, MAPK3, MAPK1, TP53, PIK3CA, and STAT3 may be potential protein targets for BYTQ to treat AR. Molecular docking analysis indicated that the active compounds of BYTQ could bind tightly to these key targets. In addition, BYTQ could inhibit OVA-induced phosphorylation levels of PI3K, AKT1, STAT3 and ERK1/2. The CETSA data suggested that BYTQ could improve the heat stability of PI3K, AKT1, STAT3 and ERK1/2. ConclusionsBYTQ suppresses E-selectin and VCAM-1 and ICAM1 expression by regulating PI3K/AKT and STAT3/MAPK signaling pathways, thus alleviating inflammation in AR mice. BYTQ is the aggressive treatment for AR.

Levels and clinical significance of the m6A methyltransferase METTL14 in patients with coronary heart disease.

To investigate the association of methyltransferase-like protein 14 (METTL14) expression with coronary heart disease (CHD). Three hundred and sixteen patients who attended Henan Provincial People's Hospital between June 2019 and February 2021 with principal symptoms of pain or tightness in the chest and who underwent coronary angiography for definitive diagnosis were enrolled. The uric acid, TG, TC, LDL-C, HDL-C, apolipoprotein A1, free fatty acid, lipoprotein a, homocysteine, CRP, and SAA levels were examined. The levels of METTL14, TNF-α, MCP-1, VCAM-1, ICAM-1, and IL-6 were evaluated by ELISA. Patients with CHD had significantly higher m6A methyltransferase activity. In addition, the incidence of diabetes and hypertension, as well as the concentrations of TC, CRP, and SAA were higher in CHD patients. Patients with coronary lesion branches also had significantly increased TG, LDL-C, CRP, and SAA levels. TNF-α, MCP-1, VCAM-1, ICAM-1, and IL-6 expression was also markedly increased in the CHD group (P < 0.001) as was the expression of METTL14 (P < 0.001). The METTL14 expression levels also differed significantly in relation to the number of branches with lesions (P < 0.01) and were correlated with SAA, VCAM-1, ICAM-1, IL-6, and the Gensini score. ROC curve analyses of METTL14 in CHD indicated an AUC of 0.881 (0.679, 0.894) with a cut-off value of 342.37, a sensitivity of 77%, and a specificity of 84%. MCP-1, VCAM-1, IL-6, SAA, and METTL14 were found to independently predict CHD risk. METTL14 levels were found to be positively associated with inflammatory markers and to be an independent predictor of CHD risk.

The Interplay between Atherosclerosis and Cancer: Breast Cancer Cells Increase the Expression of Endothelial Cell Adhesion Markers.

Cardiovascular diseases are the leading causes of death worldwide, closely followed by cancer. To investigate the impact of breast cancer cell lines (SKBR3, MCF-7, and MDA-MB-231) on endothelial cell adhesion, a blended medium containing 30% breast-cancer-conditioned medium was prepared. This medium was then exposed to human umbilical vein endothelial cells (HUVECs) and monocytes (THP-1) for 48 h. Homemade oxidized low-density lipoproteins (oxLDL) were optionally added to the blended medium. Immunofluorescence was performed to assess the expression of E-selectin, connexin-43, and ICAM-1 on HUVECs, as well as LOX-1, CD36, and CD162 on THP-1. Additionally, unoxidized LDL was exposed to the three breast cancer cell lines for 48 h, and the formation of oxLDL was quantified. Our results revealed an upregulation of all six adhesion markers involved in the initiation of atherosclerosis when HUVECs and THP-1 were exposed to the breast-cancer-conditioned medium. Furthermore, this expression was further increased by exposure to oxLDL. We also observed a significant elevation in oxLDL levels when LDL was exposed to breast cancer cells. In conclusion, our findings successfully demonstrate an increased LDL oxidation in the presence of breast cancer cells, accompanied by an augmented expression of receptors involved in atherosclerosis initiation. These findings shed new light on the clinically observed interplay between atherosclerosis and cancer.

Curcumin nanocrystals self-stabilized Pickering emulsion freeze-dried powder: Development, characterization, and suppression of airway inflammation

Curcumin, a diketone compound extracted from turmeric's rhizome, is an effective anti-inflammatory drug with multiple pharmacological activities. However, its low oral bioavailability due to its low water solubility and permeability severely limits its clinical applications. Therefore, to enhance the oral bioavailability of curcumin, further enhance its anti-inflammatory effects, and improve its potential in the treatment of airway inflammation, a curcumin nanocrystalline self-stabilizing Pickering emulsion (Cur-NSSPE) was prepared through high-pressure homogenization. Next, Cur-NSSPE was dried using a freeze-drying method to produce Cur-NSSPE-FDP. The prepared Cur-NSSPE and Cur-NSSPE-FDP were physically characterized. The release behavior and transmembrane transport capability of Cur-NSSPE-FDP in vitro were evaluated. Pharmacokinetic study was performed to evaluate its oral bioavailability. The anti-inflammatory effects of Cur-NSSPE-FDP in vivo and in vitro were investigated using RAW 264.7 macrophage inflammation model induced by LPS and IFN-γ and asthma model in BALB/c mice induced by OVA. The average particle size of Cur-NSSPE was (163.66 ± 6.78) nm, and the average drug content was (2.78 ± 0.01) mg/mL. The transmission electron microscopy results showed that the droplets were spherical in shape with a relatively uniform size, and the curcumin nanocrystals formed a spherical core-shell structure wrapped at the interface of the droplets. The scanning electron microscopy showed that Cur-NSSPE-FDP was a neatly arranged, having loose and porous network structure. Furthermore, it can significantly improve the cumulative release of curcumin in vitro and improve oral bioavailability in rats, increase the uptake of RAW264.7 and Caco-2 cells, promote the transport of curcumin across Caco-2 cells, significantly inhibit the expression of inflammatory factors NO, IL-6, TNF-a, MDA, IgE and ICAM-1, and improve the expression of IL-10 and SOD. These results indicated that the curcumin nanocrystalline self-stabilizing Pickering emulsion-freeze dried powder improved the oral bioavailability of curcumin and enhanced its therapeutic effect in airway inflammation.