Upregulation Of ICAM-1 Research Articles

Total flavone of flowers of Abelmoschus manihot (L.) Medic inhibits the expression of adhesion molecules in primary mesenteric arterial endothelial cells and ameliorates dextran sodium sulphate-induced ulcerative colitis in mice

BackgroundFlowers of Abelmoschus manihot (L.) medic (AM) is a traditional Chinese medicine used to treat chronic nephritis, nephrotic syndrome, diabetic nephropathy, and colonic inflammation. PurposeThis study aimed to explore the influence of the total flavone of AM flowers (TFA) on acute ulcerative colitis (UC) and the potential underlying mechanism. MethodsEfficacy of TFA (30, 60, 120 mg/kg) on UC was evaluated in a dextran sodium sulphate (DSS)-induced colonic inflammatory mouse model by analyzing disease activity index (DAI), histopathological score, colon length, and cytokine expression. Expression levels of critical adhesion molecules and nuclear factor kappa B (NF-κB) were examined by qRT-PCR, Western blotting, or immunofluorescence labeling. Myeloperoxidase activity was examined using ELISA. In vitro THP-1 adhesion assay was used to evaluate monocyte adhesion. ResultsTFA significantly reduced DAI score, prevented colon shortening, and ameliorated histological injuries of colons in DSS-treated mice. TFA inhibited the expression of cytokines (IL-1β and TNF-α) and adhesion molecules (ICAM-1, VCAM-1, and MAdCAM-1) in colon tissues of DSS mice. In vitro studies on mesenteric arterial endothelial cells (MAECs) showed that TFA attenuated TNF-α-induced upregulation of ICAM-1, VCAM-1, and MAdCAM-1, as well as THP-1 cell adhesion to MAECs. TFA also suppressed the phosphorylation and nuclear translocation of NF-κB in MAECs. ConclusionTFA efficaciously ameliorates UC possibly by inhibiting monocyte adhesion through blocking TNF-α-induced NF-κB activation, which in turn suppresses the upregulation of adhesive molecules in colon endothelial cells. Inhibiting the expression of adhesion molecule in MAECs may represent a useful strategy for therapeutic development to treat UC, with TFA being a safe and efficacious therapeutic agent.

Abstract WMP113: Red Blood Cells From Stroke Patients With Admission Hyperglycemia Induces Brain Endothelial Dysfunction

Background: Beyond its traditional role as oxygen carriers, red blood cells (RBCs) are increasingly recognized to participate in regulating vascular function. Many RBC disorders, such as sickle cell disease, are associated with vascular complications resulting in stroke. Our recent study revealed that admission hyperglycemia in stroke patients intensifies oxidative stress in RBCs. We hypothesize that RBCs that are altered by hyperglycemia may perturb endothelial function. Method: Brain endothelial cells were incubated with RBCs isolated from stroke patients with admission hyperglycemia (hyper-RBC) and normoglycemia (normo-RBC) (n=3/group). Mass spectrometry was used to profile the changes in endothelial cells. Functional alterations were assessed by gene set enrichment analysis (GSEA). Significant changes were verified using qPCR and western blot. Result: Compared to normo-RBC, hyper-RBC activates cell adhesion process and toll like receptor signaling in brain endothelial cells (Fig A, B). Cell adhesion molecules (ICAM1, VCAM1) and toll like receptor TLR4 were upregulated at both mRNA and protein levels (Fig C, D), which may promote vaso-occlusion and inflammation. In contrast, vasoregulation was deactivated by hyper-RBC (Fig A, B). The expression of eNOS was downregulated and the bioavailability of vasodilator NO was decreased, consistent with impaired vasodilation (Fig C, D). Notably, the changes induced by hyper-RBC could be reversed by pretreating hyper-RBC with ROS scavenger TEMPOL. The upregulation of ICAM1, VCAM1 and TLR4 in hyper-RBC-treated cells was attenuated by TEMPOL pretreatment, and eNOS expression and NO bioavailability were also restored by TEMPOL (Fig C, D). Conclusion: Intensified oxidative stress in RBCs induced by admission hyperglycemia in stroke patients can cause endothelial dysfunction. Further studies are underway to validate these targets, and ask whether these mechanisms may exacerbate brain injury in stroke patients.

E3 Ubiquitin Ligase Midline 1 Regulates Endothelial Cell ICAM-1 Expression and Neutrophil Adhesion in Abdominal Sepsis

Septic lung damage is associated with endothelial cell and neutrophil activation. This study examines the role of the E3 ubiquitin ligase midline 1 (Mid1) in abdominal sepsis. Mid1 expression was increased in endothelial cells derived from post-capillary venules in septic mice and TNF-α challenge increased Mid1 levels in endothelial cells in vitro. The siRNA-mediated knockdown of Mid1 decreased TNF-α-induced upregulation of ICAM-1 and neutrophil adhesion to endothelial cells. Moreover, Mid1 silencing reduced leukocyte adhesion in post-capillary venules in septic lungs in vivo. The silencing of Mid1 not only decreased Mid1 expression but also attenuated expression of ICAM-1 in lungs from septic mice. Lastly, TNF-α stimulation decreased PP2Ac levels in endothelial cells in vitro, which was reversed in endothelial cells pretreated with siRNA directed against Mid1. Thus, our novel data show that Mid1 is an important regulator of ICAM-1 expression and neutrophil adhesion in vitro and septic lung injury in vivo. A possible target of Mid1 is PP2Ac in endothelial cells. Targeting the Mid1-PP2Ac axis may be a useful way to reduce pathological lung inflammation in abdominal sepsis.

IL-33 Induces an Antiviral Signature in Mast Cells but Enhances Their Permissiveness for Human Rhinovirus Infection.

Mast cells (MCs) are classically associated with allergic asthma but their role in antiviral immunity is unclear. Human rhinoviruses (HRVs) are a major cause of asthma exacerbations and can infect and replicate within MCs. The primary site of HRV infection is the airway epithelium and MCs localise to this site with increasing asthma severity. The asthma susceptibility gene, IL-33, encodes an epithelial-derived cytokine released following HRV infection but its impact on MC antiviral responses has yet to be determined. In this study we investigated the global response of LAD2 MCs to IL-33 stimulation using RNA sequencing and identified genes involved in antiviral immunity. In spite of this, IL-33 treatment increased permissiveness of MCs to HRV16 infection which, from the RNA-Seq data, we attributed to upregulation of ICAM1. Flow cytometric analysis confirmed an IL-33-dependent increase in ICAM1 surface expression as well as LDLR, the receptors used by major and minor group HRVs for cellular entry. Neutralisation of ICAM1 reduced the IL-33-dependent enhancement in HRV16 replication and release in both LAD2 MCs and cord blood derived MCs. These findings demonstrate that although IL-33 induces an antiviral signature in MCs, it also upregulates the receptors for HRV entry to enhance infection. This highlights the potential for a gene-environment interaction involving IL33 and HRV in MCs to contribute to virus-induced asthma exacerbations.

Lymphatic coagulation and neutrophil extracellular traps in lung-draining lymph nodes of COVID-19 decedents

AbstractClinical manifestations of severe COVID-19 include coagulopathies that are exacerbated by the formation of neutrophil extracellular traps (NETs). Here, we report that pulmonary lymphatic vessels, which traffic neutrophils and other immune cells to the lung-draining lymph node (LDLN), can also be blocked by fibrin clots in severe COVID-19. Immunostained tissue sections from COVID-19 decedents revealed widespread lymphatic clotting not only in the lung but also in the LDLN, where the extent of clotting correlated with the presence of abnormal, regressed, or missing germinal centers (GCs). It strongly correlated with the presence of intralymphatic NETs. In mice, tumor necrosis factor α induced intralymphatic fibrin clots; this could be inhibited by DNase I, which degrades NETs. In vitro, TNF-α induced lymphatic endothelial cell upregulation of ICAM-1 and CXCL8, among other neutrophil-recruiting factors, as well as thrombomodulin downregulation; in decedents, lymphatic clotting in LDLNs. In a separate cohort of hospitalized patients, serum levels of Myeloperoxidase-DNA (MPO-DNA, a NET marker) inversely correlated with antiviral antibody titers, but D-dimer levels, indicative of blood thrombosis, did not correlate with either. Patients with high MPO-DNA but low D-dimer levels generated poor antiviral antibody titers. This study introduces lymphatic coagulation in lungs and LDLNs as a clinical manifestation of severe COVID-19 and suggests the involvement of NETosis of lymphatic-trafficking neutrophils. It further suggests that lymphatic clotting may correlate with impaired formation or maintenance of GCs necessary for robust antiviral antibody responses, although further studies are needed to determine whether and how lymphatic coagulation affects adaptive immune responses.

IL11 Stimulates IL33 Expression and Proinflammatory Fibroblast Activation across Tissues.

Interleukin 11 (IL11) is upregulated in inflammatory conditions, where it is mostly believed to have anti-inflammatory activity. However, recent studies suggest instead that IL11 promotes inflammation by activating fibroblasts. Here, we assessed whether IL11 is pro- or anti-inflammatory in fibroblasts. Primary cultures of human kidney, lung or skin fibroblasts were stimulated with IL11 that resulted in the transient phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the sustained activation of extracellular signal-regulated protein kinases (ERK). RNA sequencing over a time course of IL11 stimulation revealed a robust but short-lived transcriptional response that was enriched for gene set hallmarks of inflammation and characterized by the upregulation of SERPINB2, TNFRSF18, Interleukin 33 (IL33), CCL20, IL1RL1, CXCL3/5/8, ICAM1 and IL11 itself. IL33 was the most upregulated signaling factor (38-fold, p = 9.8 × 10−5), and IL1RL1, its cognate receptor, was similarly increased (18-fold, p = 1.1 × 10−34). In proteomic studies, IL11 triggered a proinflammatory secretome with the notable upregulation of IL8, IL6, MCP1, CCL20 and CXCL1/5/6, which are important chemotaxins for neutrophils, monocytes, and lymphocytes. IL11 induced IL33 expression across fibroblast types, and the inhibition of STAT3 but not of MEK/ERK prevented this. These data establish IL11 as pro-inflammatory with specific importance for priming the IL33 alarmin response in inflammatory fibroblasts across tissues.

P1483: EPELEUTON, A NOVEL SYNTHETIC SECOND GENERATION W-3 FATTY ACID, PROTECTS HUMANIZED SICKLE CELL MICE AGAINST HYPOXIA/REOXYGENATION ORGAN DAMAGE

Background: Sickle cell disease (SCD) is a hereditary red cell disorder with high mortality and morbidity. Although crizanlizumab, a P-selectin inhibitor, and voxelotor, an oral anti-sickling agent, have been recently approved for clinical management of SCD, therapeutic options to limit acute events and to reduce disease progression are still required. An attempt to target inflammatory vasculopathy and to modulate inflammatory response has been made based on evidence in other diseases such as in cardiovascular disease looking to administration of ω-3 fatty acids. In humanized SCD mice, we previously showed that polyunsaturated fatty acid supplementation protects against acute sickle cell-related lung and liver damages during hypoxia/reoxygenation (H/R) induced vaso-occlusive crisis (VOCs) (Kalish B et al. Haematologica 2016). This agrees with the observation that the resolution process actively controlled by specialized pro-resolving lipid mediators is defective in SCD (Matte A et al, Blood 2019). Epeleuton (15-HEPE, 15 hydroxy eicosapentaenoic acid) is a novel synthetic second generation w-3 fatty acid which (i) has a favorable clinical safety profile, (ii) modulates systemic inflammation and triglyceride synthesis in patients with cardiometabolic disease (Climax J et al J Am Heart As 2020) and (ii) beneficially affects red cell indices in long-term studies in rats. Aims: To evaluate the impact of epeleuton on acute organ damage in a humanized mouse model of SCD. Methods: 3-4 month old male and female mice healthy control (Hbatm1(HBA)Tow Hbbtm3(HBG1,HBB)Tow) and Tim Townes sickle mice (SCD, Hba tm1(HBA)Tow Hbb tm2(HBG1,HBB*)Tow) were used. Animals were treated for 6 weeks with epeleuton 1,000 mg/kg once daily or vehicle by gavage. Mice underwent 10 hours of hypoxia (8% oxygen), followed by 3 hours of reoxygenation (21% oxygen) to mimic acute VOCs (Matte A et al, Blood 2019). Hematologic parameters and molecular analysis of target organs for SCD such as lung were carried out. Results: In SCD mice exposed to H/R stress, we found that epeleuton (i) reduces H/R induced hemolysis; (ii) normalizes tyrosine-phosphorylation profile of red cell membrane proteins; and (iii) decreases neutrophil count. In lungs of SCD mice exposed to H/R stress, we observed that epeleuton prevents the H/R induced activation of inflammatory and redox related transcription factors NF-kB, p65 and Nrf2 local inflammatory response compared with vehicle-treated animals. Epeleuton treated SCD mice exposed to H/R stress showed down-regulation of VCAM-1 and E-selectin, markers of inflammatory vasculopathy as well as reduction of lung protein oxidation and expression of anti-oxidant systems such as heme-oxygenase-1 (HO-1) or peroxiredoxin-2. Similar changes were also observed in livers of epeleuton-treated SCD mice exposed to H/R stress compared with vehicle-treated SCD animals. Of note, epeleuton prevented the H/R induced up-regulation of both VCAM-1 and ICAM-1, suggesting a direct impact of treatment on vascular activation and inflammation. Summary/Conclusion: In humanized SCD mice exposed to H/R stress to mimic acute VOCs, epeleuton acts as a multimodal agent targeting red cells, hemolysis, inflammatory response, and vascular dysfunction. Our data provide the rationale for epeleuton as a potential novel therapeutic option for clinical management of patients with SCD.

Abstract 3637: Age-dependent loss of HAPLN1 erodes vascular integrity via upregulation of endothelial ICAM1 in melanoma

Abstract The 5-year survival rate for metastatic melanoma is only 23%, and patients over 55 years old have higher incidence of metastasis than younger patients independent of other prognostic factors. Our lab has shown that age-dependent changes in the tumor microenvironment drive this disparity. In particular, loss of a secreted extracellular matrix protein, HAPLN1, in the aged dermis confers increased metastasis via matrix breakdown and resulting cellular dysfunction. In this study we found that HAPLN1-mediated matrix changes impede the barrier function of blood vessels and upregulate vascular ligands which in turn increase the propensity for melanoma intravasation and metastasis via this route. VE-cadherin is a key endothelial cell junctional protein whose expression is directly correlated with vascular integrity. We showed via IHC that intradermal treatment of aged mice with recombinant HAPLN1 (rHAPLN1) is sufficient to significantly increase VE-cadherin levels in intratumoral blood vessels compared to untreated aged mice, equal to levels seen in young mice. Further, we isolated the contribution of the matrix by producing in vitro fibroblast derived matrices (FDMs) from the dermal fibroblasts of healthy young and aged donors. An impedance-based assay showed that resistance of human endothelial cell (HUVEC) monolayers is significantly decreased when cultured on aged matrix compared to young or aged+rHAPLN1 matrices. We found via qRT-PCR screen that HUVECs cultured on HAPLN1-low FDMs (aged, young+shHAPLN1) have upregulated ICAM1 compared those in HAPLN1-high environments (young, aged+rHAPLN1). ICAM1 is an endothelial cell receptor whose upregulation initiates a signaling cascade to downregulate VE-cadherin and increase vascular permeability. We confirmed the HAPLN1-dependent expression of ICAM1 in vivo via IHC of murine tumors. ICAM1 expression is known to be modulated in response to ECM stiffness. We have shown that matrices formed by aged fibroblasts are stiffer than those formed by young, and that this is due in part to the absence of HAPLN1. We created artificially stiffened FDMs via chemical cross-linking of otherwise pliable young FDMs to determine if stiffness alone was sufficient to upregulate ICAM1 on endothelial cells. We found via qRT-PCR that artificially stiffened young matrices were sufficient to increase endothelial ICAM1 levels on par with those in aged and young+shHAPLN1 conditions. This is critical, as endothelial ICAM1 is upregulated in melanoma and other cancers and is used as a ligand for cancer cells to move through the vasculature. In sum, we have found that age-dependent loss of HAPLN1 in the dermal ECM confers increased matrix stiffness which leads to the upregulation of endothelial ICAM1. This in turn increases vascular permeability to allow melanoma cells to more easily intravasate and metastasize via the hematogenous pathway in the aged system. Citation Format: Gloria E. Marino, Yash Chhabra, Amanpreet Kaur, Filipe Almeida, Ying Liu, Karin T. S. Eisinger-Mathason, Ashani T. Weeraratna. Age-dependent loss of HAPLN1 erodes vascular integrity via upregulation of endothelial ICAM1 in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3637.

Abstract 516: Splice Factor Polypyrimidine Tract-binding Protein 1 (PTBP1) Primes Endothelial Inflammation In Atherogenic Disturbed Flow Conditions

NFκB mediated endothelial activation drives leukocyte recruitment and atherosclerosis, in part through upregulation of adhesion molecules Icam1 and Vcam. The endothelium is primed for cytokine activation of NFκB by exposure to low and disturbed blood flow (LDF). While priming leads to an exaggerated expression of Icam1 and Vcam following cytokine stimulation, the molecular underpinnings are not understood. In a model of LDF, platelets were required for the increased expression of several RNA-binding splice factors, including Polypyrimidine tract binding protein (Ptbp1). This was coordinated with changes in RNA splicing in the NFκB pathway in primed cells, leading us to examine splice factors as mediators of priming. Using Icam1 and Vcam induction by TNFα stimulation as a readout, we performed a CRISPR-Cas9 screen of these factors and identified a requirement for Ptbp1 in priming. Deletion of Ptbp1 had no obvious effect on cell proliferation or viability, but reversed LDF splicing patterns and inhibited NFκB nuclear translocation and transcriptional activation of nearly all downstream targets, including Icam1 and Vcam. In human coronary arteries, elevated PTBP1 correlates with expression of TNF pathway genes and plaque. In vivo , endothelial specific deletion of Ptbp1 reduced Icam1 expression and myeloid cell infiltration at regions of LDF in atherosclerotic mice and limited atherosclerosis. Together, our data show that Ptbp1, which is induced in a subset of the endothelium by innate immune cell recruitment at regions of LDF, is required for priming of the endothelium for subsequent NFκB activation, myeloid cell recruitment and atherosclerosis.

Mechanistic Insights into Macrophages Regulation of Neutrophil Transendothelial Migration in Inflamed Mucosa

Rapid neutrophil (PMN) mobilization to sites of insult is critical for host defense and requires crossing of the vascular wall. PMN transendothelial migration (TEM) involves several well‐studied sequential adhesive interactions with vascular ECs, however what initiates or terminates this process is not well‐understood. Our findings identified a new mechanism where gut interstitial macrophages, which are rapidly recruited towards the vascular wall in response to inflammatory cues, were found to locally prime endothelial cells (ECs) responses to regulate PMN TEM. Using real‐time intravital microscopy (IVM) on lipopolysaccharides (LPS)‐inflamed intestines in anesthetized CX3CR1‐EGFP macrophage‐reporter mice, complemented by whole‐mount tissue imaging we demonstrate that macrophage presence was critical for the induction of PMN‐ECs adhesive interactions and subsequent PMN recruitment and accumulation in the intestinal mucosa. Anti CSFR‐1 antibody‐based macrophage depletion in the lamina propria and at the vessel wall significantly reduced PMN adhesion and TEM in inflamed intestines. We further observed that macrophages at the vessel wall localized specifically to regions of high ICAM‐1 intensity and their removal resulted in elimination of the ICAM‐1 “hot spots”, overall lowering the ECs ICAM‐1 expression. Mechanistically, using murine/human ECs‐macrophage and PMN co‐cultures we established that activated macrophages elevate PMN adhesion and TEM via TNFα‐dependent upregulation of ECs ICAM‐1. Antibody‐mediated neutralization of TNFα in macrophage co‐cultures with ECs and/or PMNs suppressed ICAM‐1 upregulation, decreasing PMN TEM. Further in vivo imaging studies of inflamed gut revealed high TNFα expression in macrophages and specific expression of TNFa receptor type II (TNFR II) but not type I in intestinal ECs. The use of bone marrow chimeras with TNFα knockout macrophages further confirmed the novel role of macrophages‐TNFα in regulating ECs adhesion molecule expression and PMN TEM. As such, our findings identify new, clinically relevant mechanism by which macrophages regulate PMN trafficking in inflamed mucosa.

Fusobacterium nucleatum promotes colorectal cancer cells adhesion to endothelial cells and facilitates extravasation and metastasis by inducing ALPK1/NF-κB/ICAM1 axis

ABSTRACT Metastasis is the leading cause of death for colorectal cancer (CRC) patients, and the spreading tumor cells adhesion to endothelial cells is a critical step for extravasation and further distant metastasis. Previous studies have documented the important roles of gut microbiota-host interactions in the CRC malignancy, and Fusobacterium nucleatum (F. nucleatum) was reported to increase proliferation and invasive activities of CRC cells. However, the potential functions and underlying mechanisms of F. nucleatum in the interactions between CRC cells and endothelial cells and subsequent extravasation remain unclear. Here, we uncovered that F. nucleatum enhanced the adhesion of CRC cells to endothelial cells, promoted extravasation and metastasis by inducing ICAM1 expression. Mechanistically, we identified that F. nucleatum induced a new pattern recognition receptor ALPK1 to activate NF-κB pathway, resulting in the upregulation of ICAM1. Interestingly, the abundance of F. nucleatum in tumor tissues of CRC patients was positively associated with the expression levels of ALPK1 and ICAM1. Moreover, high expression of ALPK1 or ICAM1 was significantly associated with a shorter overall survival time of CRC patients. This study provides a new insight into the role of gut microbiota in engaging into the distant metastasis of CRC cells.

Ageing and interferon gamma response drive the phenotype of neutrophils in the inflamed joint

ObjectiveNeutrophils are typically the most abundant leucocyte in arthritic synovial fluid. We sought to understand changes that occur in neutrophils as they migrate from blood to joint.MethodsWe performed RNA sequencing...

Tumor-Derived PGRN Inhibits Cd8 <sup>+</sup> T Cell Recruitment and Promotes the Progression of Breast Cancer by Up-Regulation of ICAM-1 on TAM

Tumor-Derived PGRN Inhibits Cd8 <sup>+</sup> T Cell Recruitment and Promotes the Progression of Breast Cancer by Up-Regulation of ICAM-1 on TAM

Plasma-derived exosomes implement miR-126-associated regulation of cytokines secretion in PBMCs of CHF patients in vitro.

Background and aimThe investigation of regulatory effects of intra-exosomal compounds, especially microRNAs, has promising therapeutic prospects in the treatment of numerous diseases, including cardiovascular disorders. In this study, we investigated the effect of healthy donors plasma exosomes (HDPE) on the production of cytokines by PBMC cells of patients with congestive heart failure (CHF) and showed the integral role of miRNA-126 in CHF-mediated changes of mononuclear paracrine secretion.MethodsPeripheral blood mononuclear cells (PBMСs) were isolated from a peripheral blood of fifteen patients with CHF (age, 66,8 ± 9,8 years; left ventricular ejection fraction, 44 ± 19%). The concentration of cytokines (IL-10, ICAM-1, VEGF-A, TNF-α and MCP-1) in culture medium and PBMCs was measured by ELISA. The level of miRNA-126 expression in PBMCs was performed by real-time PCR.Results:Dramatic increase of ICAM-1 level in activated PBMCs of CHF patients, as well as an increase of the IL-10, ICAM-1 and TNF-α levels in the culture medium was observed. It was accompanied by CHF-related miRNA-126 overexpression in PBMCs. HDPE treatment distinguished by a tendency to reduction in miRNA-126 expression by CHF PBMCs and correlated with upregulation of IL-10, ICAM-1, TNF-α and MCP-1 with normalization of cytokines secretion.Conclusions:The altered paracrine secretion of cytokines by CHF PBMCs and miRNA-126 overexpression in vitro was found. HDPE treatment modulated production and secretion of most of studied cytokines by CHF PBMCs in vitro. The experimental application of exosomes for the modulation of paracrine secretion and PBMCs cellular functions may be promising for CVD therapy, including endothelial dysfunction and CHF. (www.actabiomedica.it)

CAR T-cell Entry into Tumor Islets Is a Two-Step Process Dependent on IFNγ and ICAM-1.

Adoptive transfer of T cells expressing chimeric antigen receptors (CAR) has shown remarkable clinical efficacy against advanced B-cell malignancies but not yet against solid tumors. Here, we used fluorescent imaging microscopy and ex vivo assays to compare the early functional responses (migration, Ca2+, and cytotoxicity) of CD20 and EGFR CAR T cells upon contact with malignant B cells and carcinoma cells. Our results indicated that CD20 CAR T cells rapidly form productive ICAM-1-dependent conjugates with their targets. By comparison, EGFR CAR T cells only initially interacted with a subset of carcinoma cells located at the periphery of tumor islets. After this initial peripheral activation, EGFR CAR T cells progressively relocated to the center of tumor cell regions. The analysis of this two-step entry process showed that activated CAR T cells triggered the upregulation of ICAM-1 on tumor cells in an IFNγ-dependent pathway. The ICAM-1/LFA-1 interaction interference, through antibody or shRNA blockade, prevented CAR T-cell enrichment in tumor islets. The requirement for IFNγ and ICAM-1 to enable CAR T-cell entry into tumor islets is of significance for improving CAR T-cell therapy in solid tumors.

NOX1 mediates metabolic heart disease in mice and is upregulated in monocytes of humans with diastolic dysfunction.

Microvascular inflammation plays an important role in the pathogenesis of diastolic dysfunction (DD) and metabolic heart disease. NOX1 is expressed in vascular and immune cells and has been implicated in the vascular pathology of metabolic disease. However, its contribution to metabolic heart disease is less understood. NOX1-deficient mice (KO) and male wild-type (WT) littermates were fed a high-fat high-sucrose diet (HFHS) and injected streptozotocin (75 mg/kg i.p.) or control diet (CTD) and sodium citrate. Despite similar weight gain and increase in fasting blood glucose and insulin, only WT-HFHS but not KO-HFHS mice developed concentric cardiac hypertrophy and elevated left ventricular filling pressure. This was associated with increased endothelial adhesion molecule expression, accumulation of Mac-2-, IL-1β-, and NLRP3-positive cells and nitrosative stress in WT-HFHS but not KO-HFHS hearts. Nox1 mRNA was solidly expressed in CD45+ immune cells isolated from healthy mouse hearts but was negligible in cardiac CD31+ endothelial cells. However, in vitro, Nox1 expression increased in response to lipopolysaccharide (LPS) in endothelial cells and contributed to LPS-induced upregulation of Icam-1. Nox1 was also upregulated in mouse bone marrow-derived macrophages in response to LPS. In peripheral monocytes from age- and sex-matched symptomatic patients with and without DD, NOX1 was significantly higher in patients with DD compared to those without DD. NOX1 mediates endothelial activation and contributes to myocardial inflammation and remodelling in metabolic disease in mice. Given its high expression in monocytes of humans with DD, NOX1 may represent a potential target to mitigate heart disease associated with DD.

Doxorubicin-Resistant TNBC Cells Exhibit Rapid Growth with Cancer Stem Cell-like Properties and EMT Phenotype, Which Can Be Transferred to Parental Cells through Autocrine Signaling.

Emerging evidence suggests that breast cancer stem cells (BCSCs), and epithelial–mesenchymal transition (EMT) may be involved in resistance to doxorubicin. However, it is unlear whether the doxorubicin-induced EMT and expansion of BCSCs is related to cancer dormancy, or outgrowing cancer cells with maintaining resistance to doxorubicin, or whether the phenotypes can be transferred to other doxorubicin-sensitive cells. Here, we characterized the phenotype of doxorubicin-resistant TNBC cells while monitoring the EMT process and expansion of CSCs during the establishment of doxorubicin-resistant MDA-MB-231 human breast cancer cells (DRM cells). In addition, we assessed the potential signaling associated with the EMT process and expansion of CSCs in doxorubicin-resistance of DRM cells. DRM cells exhibited morphological changes from spindle-shaped MDA-MB-231 cells into round-shaped giant cells. They exhibited highly proliferative, EMT, adhesive, and invasive phenotypes. Molecularly, they showed up-regulation of Cyclin D1, mesenchymal markers (β-catenin, and N-cadherin), MMP-2, MMP-9, ICAM-1 and down-regulation of E-cadherin. As the molecular mechanisms responsible for the resistance to doxorubicin, up-regulation of EGFR and its downstream signaling, were suggested. AKT and ERK1/2 expression were also increased in DRM cells with the advancement of resistance to doxorubicin. Furthermore, doxorubicin resistance of DRM cells can be transferred by autocrine signaling. In conclusion, DRM cells harbored EMT features with CSC properties possessing increased proliferation, invasion, migration, and adhesion ability. The doxorubicin resistance, and doxorubicin-induced EMT and CSC properties of DRM cells, can be transferred to parental cells through autocrine signaling. Lastly, this feature of DRM cells might be associated with the up-regulation of EGFR.

Abstract 14129: S18 Alleviates The Inflammatory Responses In Interstitial Cells Of Aortic Valves Through Up-regulation Interleukin 37

Background: Calcific aortic valve disease (CAVD) is a common cardiovascular disease,which leads to the failure of valvular function in the elderly. CAVD is characterized by complex pathophysiological processes, including inflammation-induced osteoblastic differentiation of aortic valve interstitial cells (AVICs). Anti-CAVD agents with new mechanisms of action are urgently needed. S18 is obtained from a marine-derived Streptomyces strain, and it has a potent anti-inflammatory effect. However, its effect on CAVD is unknown. This study is sought to investigate the role of the natural product S18 in alleviating CAVD progression. Methods and Results: Human AVICs treated with LPS showed up-regulation of inflammatory factors ICAM-1, IL-8, and MCP-1, and significant calcification evaluated by Alizarin Red staining. S18 (2 μM) attenuated LPS-induced human AVICs inflammation. RNA-sequencing was used to identify differentially expressed genes (DEGs) in human AVICs treated with LPS or LPS + S18. Transcriptome data indicated the common DEGs enrichment in NF-κB pathway. Further, S18 inhibited phosphorylation of NF-κB induced by LPS. Previous studies suggested that IL-37 is a potential anti-inflammatory target of S18 in AVICs. Knockdown of IL-37 by siRNA exaggerated LPS-induced inflammation, and attenuated the anti-inflammatory effect of S18 on AVICs. Conclusion: Our results demonstrate that S18 attenuates LPS-mediated inflammation in human AVICs through inhibiting NF-κB activation, and underlying the mechanism of the anti-inflammatory effect involving up-regulation of IL-37. Our finding provides a novel avenue of preventing the progression of valve calcification.

Abstract 11334: Metabolic Labeling and Systemic Tracking of Cardiomyocyte-Derived Exosomal MiRNAs

Background & Hypothesis: Following acute myocardial infarction (AMI), cardiac exosomes are released into circulation ( PB Exo), thereby transferring cargo miRNAs (miRs) to remote organs to trigger systemic responses. However, the scope and impact of cardiac miR transfer is largely unknown. Since T. gondii uracil phosphoribosyltransferase (UPRT) can convert 4-thiouracil into 4-thiouridine (4TU), then incorporating into nascent RNAs, and the 4TU-labeled RNAs can be identified by thio-linked-biotinylation and streptavidin-affinity isolation, we hypothesize that cardiomyocyte (CM)-specific expression of UPRT ( CM UPRT) allows labeling and tracking of CM-derived miRs in PB Exo and peripheral organs. Methods & Results: We generated CM UPRT mice by crossing αMHC-Cre and CA-GFPstop flox - UPRT mice. Six hours after 4-thiouracil injection, RNAs were isolated from the heart and PB Exo and found to be labeled with high specificity and sensitivity. Then we attempted to isolate representative miRs of different tissue specificities from PB Exo and multiple organs; only CM specific miR-208a, but not other-tissue specific miRs, was 4TU labeled, thus isolated, abundantly in PB Exo and lung. Among FACS-sorted lung cells, endothelial cells (ECs) have the highest CM miR-208a level. Next, we monitored CM miR-208a levels after AMI, which peaked in PB Exo at 12 h and in the lung at 24 h. Since miR-208a is from the intron 29 of the cardiac α-MHC, we measured α-MHC transcript in the lung, which was barely detectable. To determine the biological effect of PB Exo-mediated CM miR-208a transfer, we i.v. injected PB Exo obtained from Sham and AMI mice to intact mice; Sham-Exo and to a greater extent, AMI-Exo, but not Exo from LNA-anti-miR-208a pre-treated mice, downregulated the protein expression of miR-208a target genes (Glyr1, Tmbim6, and NLK) in the lung. Since all the three proteins suppress key effectors in the NF-kB pathway, we examined NF-kB’s classic targets (ICAM-1, VCAM-1 and E-selectin); PB Exo-mediated Glyr1, Tmbim6, and NLK downregulation was associated with ICAM-1, VCAM-1 and E-selectin upregulation. Conclusion: CM UPRT expression permits labeling and tracking of CM miRs in PB Exo and organs; cardiac Exo and CM miR-208a may contribute to AMI-associated lung inflammation.

Monocyte Maturation Mediators Upregulate CD83, ICAM-1 and MHC Class 1 Expression on Ewing's Sarcoma, Enhancing T Cell Cytotoxicity.

Ewing’s sarcoma (EwS) is a pediatric solid tumor entity with low somatic mutational burden and a low rate of tumor-infiltrating T cells, indicating a low extent of immunogenicity. In EwS, immunogenicity may furthermore be significantly diminished by a predominantly M2 macrophage driven pro-tumorigenic tumor microenvironment. In the past, we demonstrated that CHM1319-specific TCR-transgenic T cells are able to control EwS growth in a preclinical mouse model as well as in a patient with metastatic disease. However, new adjuvant techniques to induce long lasting and curative CHM1319-specific TCR-transgenic T cell-mediated anti-tumor responses are needed. In this work, we sought to identify a technique to improve the cytotoxic effect of CHM1319-specific TCR-transgenic T cell by altering the immunogenic cell surface marker expression on EwS cell lines using different cytokines. We demonstrate that TNF, IL-6, IL-1β and PGE2 cause pro-immunogenic CD83, MHC class I and II as well as ICAM-1 upregulation in EwS cell lines. This observation was associated with significantly improved recognition and killing of the tumor cells by EwS-specific CHM1319/HLA-A*02:01-restricted TCR-transgenic T cells. Conclusively, we demonstrate that the induction of an inflammatory signature renders EwS more susceptible to adoptive T cell therapy. TNF, which is upregulated during inflammatory processes, is of particular translational interest as its secretion may be induced in the patients e.g., by irradiation and hyperthermia in the clinical setting. In future clinical protocols, this finding may be important to identify appropriate conditioning regimens as well as point of time for adoptive T cell-based immunotherapy in EwS patients.