Reactive oxygen species in endothelial signaling in COVID-19: Protective role of the novel peptide PIP-2.

INTRODUCTION: Recent research suggests that endothelial activation plays a role in coronavirus disease 2019 (COVID-19) pathogenesis by promoting a pro-inflammatory state. However, the mechanism by which the endothelium is activated in COVID-19 remains unclear. OBJECTIVE: To investigate the mechanism by which COVID-19 activates the pulmonary endothelium and drives pro-inflammatory phenotypes. HYPOTHESIS: The "inflammatory load or burden" (cytokine storm) of the systemic circulation activates endothelial NADPH oxidase 2 (NOX2) which leads to the production of reactive oxygen species (ROS) by the pulmonary endothelium. Endothelial ROS subsequently activates pro-inflammatory pathways. METHODS: The inflammatory burden of COVID-19 on the endothelial network, was recreated in vitro, by exposing human pulmonary microvascular endothelial cells (HPMVEC) to media supplemented with serum from COVID-19 affected individuals (sera were acquired from patients with COVID-19 infection that eventually died. Sera was isolated from blood collected at admission to the Intensive Care Unit of the Hospital of the University of Pennsylvania). Endothelial activation, inflammation and cell death were assessed in HPMVEC treated with serum either from patients with COVID-19 or from healthy individuals. Activation was monitored by measuring NOX2 activation (Rac1 translocation) and ROS production; inflammation (or appearance of a pro-inflammatory phenotype) was monitored by measuring the induction of moieties such as intercellular adhesion molecule (ICAM-1), P-selectin and the NLRP3 inflammasome; cell death was measured via SYTOX™ Green assays. RESULTS: Endothelial activation (i.e., NOX2 activation and subsequent ROS production) and cell death were significantly higher in the COVID-19 model than in healthy samples. When HPMVEC were pre-treated with the novel peptide PIP-2, which blocks NOX2 activation (via inhibition of Ca2+-independent phospholipase A2, aiPLA2), significant abrogation of ROS was observed. Endothelial inflammation and cell death were also significantly blunted. CONCLUSIONS: The endothelium is activated during COVID-19 via cytokine storm-driven NOX2-ROS activation, which causes a pro-inflammatory phenotype. The concept of endothelial NOX2-ROS production as a unifying pathophysiological axis in COVID-19 raises the possibility of using PIP-2 to maintain vascular health.

Unveiling the crucial role of intercellular adhesion molecule-1 in secondary diabetic complications.

Diabetes mellitus is associated with secondary complications such as diabetic retinopathy (DR), nephropathy (DN), and cardiomyopathy (DCM), all of which significantly impact patient health. Intercellular adhesion molecule-1 (ICAM-1) has been implicated in inflammatory responses and endothelial dysfunction, both crucial in the pathogenesis of these complications. The goal of this review is to investigate at potential therapy methods that target ICAM-1 pathways and to better understand the multifaceted role of ICAM-1 in secondary diabetic problems. A meticulous analysis of scholarly literature published globally was conducted to examine ICAM-1involvement in inflammatory processes, endothelial dysfunction, and oxidative stress related to diabetes and its complications. Elevated ICAM-1 levels are strongly associated with augmented leukocyte adhesion, compromised microvascular function, and heightened oxidative stress in diabetes. These pathways contribute significantly to DR, DN, and DCM pathogenesis, highlighting ICAM-1 as a key player in their progression. Understanding ICAM-1 role in secondary diabetic complications offers insights into novel therapeutic strategies. Targeting ICAM-1 pathways may mitigate inflammation, improve endothelial function, and ultimately attenuate diabetic complications, thereby enhancing patient health outcomes. Continued research in this area is crucial for developing effective targeted therapies.

miR-486-5p protects against rat ischemic kidney injury and prevents the transition to chronic kidney disease and vascular dysfunction.

AIM: Acute kidney injury (AKI) increases the risk for progressive chronic kidney disease (CKD). MicroRNA (miR)-486-5p protects against kidney ischemia-reperfusion (IR) injury in mice, although its long-term effects on the vasculature and development of CKD are unknown. We studied whether miR-486-5p would prevent the AKI to CKD transition in rat, and affect vascular function. METHODS: Adult male rats were subjected to bilateral kidney IR followed by i.v. injection of liposomal-packaged miR-486-5p (0.5 mg/kg). Kidney function and histologic injury were assessed after 24 h and 10 weeks. Kidney endothelial protein levels were measured by immunoblot and immunofluorescence, and mesenteric artery reactivity was determined by wire myography. RESULTS: In rats with IR, miR-486-5p blocked kidney endothelial cell increases in intercellular adhesion molecule-1 (ICAM-1), reduced neutrophil infiltration and histologic injury, and normalized plasma creatinine (P<0.001). However, miR-486-5p attenuated IR-induced kidney endothelial nitric oxide synthase (eNOS) expression (P<0.05). At 10 weeks, kidneys from rats with IR alone had decreased peritubular capillary density and increased interstitial collagen deposition (P<0.0001), and mesenteric arteries showed impaired endothelium-dependent vasorelaxation (P<0.001). These changes were inhibited by miR-486-5p. Delayed miR-486-5p administration (96 h, 3 weeks after IR) had no impact on kidney fibrosis, capillary density, or endothelial function. CONCLUSION: In rats, administration of miR-486-5p early after kidney IR prevents injury, and protects against CKD development and systemic endothelial dysfunction. These protective effects are associated with inhibition of endothelial ICAM-1 and occur despite reduction in eNOS. miR-486-5p holds promise for the prevention of ischemic AKI and its complications.

Unraveling the Impact of Extracellular Vesicle-Depleted Serum on Endothelial Cell Characteristics over Time.

Extracellular vesicles (EVs) are produced by all kinds of cells, including endothelial cells. It has been observed that EVs present in fetal bovine serum (FBS), broadly used in cell culture, can be a confounding factor and lead to misinterpretation of results. To investigate this phenomenon, human brain microvascular endothelial cells (HBMECs) were cultured for 2 or 24 h in the presence of EV-depleted FBS (EVdS). Cell death, gene and protein expression, and the presence of EVs isolated from these cells were evaluated. The uptake of EVs, intercellular adhesion molecule 1 (ICAM-1) expression, and monocyte adhesion to endothelial cells exposed to EVs were also evaluated. Our results revealed higher apoptosis rates in cells cultured with EVdS for 2 and 24 h. There was an increase in interleukin 8 (IL8) expression after 2 h and a decrease in interleukin 6 (IL6) and IL8 expression after 24 h of culture. Among the proteins identified in EVs isolated from cells cultured for 2 h (EV2h), several were related to ribosomes and carbon metabolism. EVs from cells cultured for 24 h (EV24h) presented a protein profile associated with cell adhesion and platelet activation. Additionally, HBMECs exhibited increased uptake of EV2h. Treatment of endothelial cells with EV2h resulted in greater ICAM-1 expression and greater adherence to monocytes than did treatment with EV24h. According to our data, HBMEC cultivated with EVdS produce EVs with different physical characteristics and protein levels that vary over time.

The heavy subunit of ferritin stimulates NLRP3 inflammasomes in hepatic stellate cells through ICAM-1 to drive hepatic inflammation.

Serum ferritin concentrations increase during hepatic inflammation and correlate with the severity of chronic liver disease. Here, we report a molecular mechanism whereby the heavy subunit of ferritin (FTH) contributes to hepatic inflammation. We found that FTH induced activation of the NLRP3 inflammasome and secretion of the proinflammatory cytokine interleukin-1ß (IL-1ß) in primary rat hepatic stellate cells (HSCs) through intercellular adhesion molecule-1 (ICAM-1). FTH-ICAM-1 stimulated the expression of Il1b, NLRP3 inflammasome activation, and the processing and secretion of IL-1ß in a manner that depended on plasma membrane remodeling, clathrin-mediated endocytosis, and lysosomal destabilization. FTH-ICAM-1 signaling at early endosomes stimulated Il1b expression, implying that this endosomal signaling primed inflammasome activation in HSCs. In contrast, lysosomal destabilization was required for FTH-induced IL-1ß secretion, suggesting that lysosomal damage activated inflammasomes. FTH induced IL-1ß production in liver slices from wild-type mice but not in those from Icam1-/- or Nlrp3-/- mice. Thus, FTH signals through its receptor ICAM-1 on HSCs to activate the NLRP3 inflammasome. We speculate that this pathway contributes to hepatic inflammation, a key process that stimulates hepatic fibrogenesis associated with chronic liver disease.

ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin.

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

Quercetin attenuates inflammation in rosacea by directly targeting p65 and ICAM-1.

AIMS: Rosacea is an inflammatory skin disease with immune and vascular dysfunction. Although there are multiple treatment strategies for rosacea, the clinical outcomes are unsatisfactory. MAIN METHODS: Combining transcriptome data and the Connectivity Map database quercetin was identified as a novel candidate for rosacea. Next, the therapeutic efficacy of quercetin was substantiated through proteomic analyses, in vivo experiments, and in vitro assays. Additionally, the utilization of DARTS, molecular docking and experimental verification revealed the therapeutic mechanisms of quercetin. KEY FINDINGS: Treatment with quercetin resulted in the following effects: (i) it effectively ameliorated rosacea-like features by reducing immune infiltration and angiogenesis; (ii) it suppressed the expression of inflammatory mediators in HaCaT cells and HDMECs; (iii) it interacted with p65 and ICAM-1 directly, and this interaction resulted in the repression of NF-κB signal and ICAM-1 expression in rosacea. SIGNIFICANCE: We show for the first time that quercetin interacted with p65 and ICAM-1 directly to alleviated inflammatory and vascular dysfunction, suggesting quercetin is a novel, promising therapeutic candidate for rosacea.

Activation of heme oxygenase-1 by laminar shear stress ameliorates high glucose-induced endothelial cell and smooth muscle cell dysfunction.

High glucose (HG)-induced endothelial cell (EC) and smooth muscle cell (SMC) dysfunction is critical in diabetes-associated atherosclerosis. However, the roles of heme oxygenase-1 (HO-1), a stress-response protein, in hemodynamic force-generated shear stress and HG-induced metabolic stress remain unclear. This investigation examined the cellular effects and mechanisms of HO-1 under physiologically high shear stress (HSS) in HG-treated ECs and adjacent SMCs. We found that exposure of human aortic ECs to HSS significantly increased HO-1 expression; however, this upregulation appeared to be independent of adenosine monophosphate-activated protein kinase, a regulator of HO-1. Furthermore, HSS inhibited the expression of HG-induced intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and reactive oxygen species (ROS) production in ECs. In an EC/SMC co-culture, compared with static conditions, subjecting ECs close to SMCs to HSS and HG significantly suppressed SMC proliferation while increasing the expression of physiological contractile phenotype markers, such as α-smooth muscle actin and serum response factor. Moreover, HSS and HG decreased the expression of vimentin, an atherogenic synthetic phenotypic marker, in SMCs. Transfecting ECs with HO-1-specific small interfering (si)RNA reversed HSS inhibition on HG-induced inflammation and ROS production in ECs. Similarly, reversed HSS inhibition on HG-induced proliferation and synthetic phenotype formation were observed in co-cultured SMCs. Our findings provide insights into the mechanisms underlying EC-SMC interplay during HG-induced metabolic stress. Strategies to promote HSS in the vessel wall, such as continuous exercise, or the development of HO-1 analogs and mimics of the HSS effect, could provide an effective approach for preventing and treating diabetes-related atherosclerotic vascular complications.

Accumulation of γδT cells in the decidua is promoted by RANKL which upregulates ICAM-1 via NF-κB.

In brief: The mechanism underlying the accumulation of γδT cells in the decidua, which helps maintain maternal-fetal immunotolerance in early pregnancy, is unknown. This study reveals that DSC-derived RANKL upregulates ICAM-1 expression via the NF-κB pathway to enable γδT cell accumulation in the early decidua. Abstract: Decidual γδT (dγδT) cells help maintain maternal-fetal immunotolerance in early pregnancy. However, the mechanism underlying the accumulation of γδT cells in the decidua is unknown. Previous work showed that RANKL upregulated intercellular adhesion molecule 1 (ICAM-1) in decidual stromal cells (DSCs), and Rankl knockout mice had limited dγδT cell populations. In this study, we measured the expression levels of RANKL/RANK and ICAM-1 in DSCs, in addition to the integrins of ICAM-1 on dγδT cells, and the number of dγδT cells from patients with recurrent spontaneous abortion (RSA) and normal pregnant women in the first trimester. RSA patients showed significantly decreased RANKL/RANK and ICAM-1/CD11a signaling in decidua, and a decreased percentage of dγδT cells, which was positively correlated with DSC-derived RANKL and ICAM-1. Next, an in vitro adhesion experiment showed that the enhanced attraction of human DSCs to dγδT cells after RANKL overexpression was almost completely aborted by anti-ICAM-1. Furthermore, Rankl knockout mice showed a significant reduction in NF-κB activity compared with wild-type controls. Finally, we applied a selective NF-κB inhibitor named PDTC to validate the role of NF-κB in RANKL-mediated ICAM-1 upregulation. Taken together, our data show that DSC-derived RANKL upregulates ICAM-1 expression via the NF-κB pathway to enable γδT cell accumulation in the early decidua. A reduction in RANKL/ICAM-1 signaling in DSCs may result in insufficient accumulation of γδT cells in decidua and, in turn, RSA.

Anti-Atherosclerotic Properties of Aronia melanocarpa Extracts Influenced by Their Chemical Composition Associated with the Ripening Stage of the Berries.

The high content of bioactive compounds in Aronia melanocarpa fruit offers health benefits. In this study, the anti-atherosclerotic effect of Aronia extracts was assessed. The impact on the level of adhesion molecules and the inflammatory response of human umbilical vein endothelial cells (HUVECs) was shown in relation to the chemical composition and the stage of ripening of the fruits. Samples were collected between May (green, unripe) and October (red, overripe) on two farms in Poland, which differed in climate. The content of chlorogenic acids, anthocyanins, and carbohydrates in the extracts was determined using HPLC-DAD/RI. The surface expression of ICAM-1 and VCAM-1 in HUVECs was determined by flow cytometry. The mRNA levels of VCAM-1, ICAM-1, IL-6, and MCP-1 were assessed using the quantitative real-time PCR method. The farms' geographical location was associated with the quantity of active compounds in berries and their anti-atherosclerotic properties. Confirmed activity for green fruits was linked to their high chlorogenic acid content.

[Neutrophil extracellular traps extrusion from neutrophils stably adhered to ICAM-1 by lipoteichoic acid stimulation].

The effect of neutrophil extracellular traps (NETs) on promoting intravascular microthrombi formation and exacerbating the severity of sepsis in patients has gained extensive attention. However, in sepsis, the mechanisms and key signaling molecules mediating NET formation during direct interactions of endothelial cells and neutrophils still need further explored. Herein, we utilized lipoteichoic acid (LTA), a component shared by Gram-positive bacteria, to induce NET extrusion from neutrophils firmly adhered to the glass slides coated with intercellular adhesion molecule-1(ICAM-1). We also used Sytox green to label NET-DNA and Flou-4 AM as the intracellular Ca 2+ signaling indicator to observe the NET formation and fluctuation of Ca 2+ signaling. Our results illustrated that LTA was able to induce NET release from neutrophils firmly attached to ICAM-1-coated glass slides, and the process was time-dependent. In addition, our study indicated that LTA-induced NET release by neutrophils stably adhered to ICAM-1 depended on Ca 2+ signaling but not intracellular reactive oxygen species (ROS). This study reveals NET formation mediated by direct interactions between endothelial ICAM-1 and neutrophils under LTA stimulation and key signaling molecules involved, providing the theoretical basis for medicine development and clinical treatment for related diseases.

Impact of TNF and IL-33 Cytokines on Mast Cells in Neuroinflammation.

Mast cells (MCs) are derived from hematopoietic progenitors, mature in vascularized tissues, and participate in innate and acquired immunity. Neuroinflammation is a highly debated topic in the biomedical literature; however, the impact of tumor necrosis factor (TNF) and IL-33 on MCs in the brain has not been widely addressed. MCs can be activated by IgE binding to FcεRI, as well as by different antigens. After activation, MCs mediate various immunological and inflammatory responses through TNF and IL-33. TNF has two receptors: TNFR1, a p55 molecule, and TNFR2, a p75 molecule. This cytokine is the only one of its kind to be stored in the granules of MCs and can also be generated by de novo synthesis via mRNA. In the central nervous system (CNS), TNF is produced almost exclusively by microglial cells, neurons, astrocytes, and, minimally, by endothelial cells. After its release into brain tissue, TNF rapidly induces the adhesion molecules endothelial leukocyte adhesion molecule 1 (ELAM-1), intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1) in endothelial cells. TNF causes the chemoattraction of neutrophils by inducing several molecules, including CXC chemokines (IL-8). Both MCs and microglial cells act as a primary barrier against foreign molecules in the CNS, producing pro-inflammatory cytokines such as IL-33. IL-33 belongs to the IL-1 family, is activated through the ST2L/IL1-RAcP receptor complex, and mediates both the innate and adaptive immune response. IL-33 is a nuclear transcription factor expressed in the brain, where it induces pro-inflammatory cytokines (TNF and IL-1) and chemokines (CCL2, CCL3, CCL5, and CXCL10). Therefore, MCs and microglia in the CNS are a source of pro-inflammatory cytokines, including TNF and IL-33, that mediate many brain diseases. The inhibition of TNF and IL-33 may represent a new therapeutic approach that could complement existing neuroinflammatory therapies.

PGRN inhibits CD8+T cell recruitment and promotes breast cancer progression by up-regulating ICAM-1 on TAM.

BACKGROUND: Tumor microenvironment actually reduces antitumor effect against the immune attack by exclusion of CD8+T cells. Progranulin (PGRN) is a multifunctional growth factor with significant pathological effects in multiple tumors; however, its role in immunity evasion of breast cancer (BCa) is not completely understood. METHODS: We depleted GRN (PGRN gene) genetically in mice or specifically in PY8119 murine BCa cell line, and mouse models of orthotopic or subcutaneous transplantation were used. Chimeric mice-deficient of PGRN (Grn-/-) in bone marrow (BM) compartment was also generated. Association of PGRN expression with chemokine production or BCa development was investigated by histological and immunological assays. RESULTS: We found PGRN was involved in exhaustion of cytotoxic CD8+T cell in BCa with the increasing expressions of M2 markers and intercellular cell adhesion molecule-1 (ICAM-1) on macrophages. Specifically, ablation of PGRN in PY8119 cells reduced tumor burden, accompanied by the infiltrating of cytotoxic CD8+T cells into tumor nests. Moreover, our result revealed that blockade of PD-1 in PGRN-depleted tumors exhibited better antitumor effect in vivo and significantly decreased tumor burden. CONCLUSION: These findings suggest that inhibition of PGRN may act as a potential immune-therapeutic strategy by recovering infiltration of CD8+T cell in BCa tissue and thereby enhancing the response to anti-PD-1 therapy.

Proteomics on human cerebral cavernous malformations reveals novel biomarkers in neurovascular dysfunction for the disease pathology.

BACKGROUND: Cerebral cavernous malformation (CCM) is a disease associated with an elevated risk of focal neurological deficits, seizures, and hemorrhagic stroke. The disease has an inflammatory profile and improved knowledge of CCM pathology mechanisms and exploration of candidate biomarkers will enable new non-invasive treatments. METHODS: We analyzed protein signatures in human CCM tissue samples by using a highly specific and sensitive multiplexing technique, proximity extension assay. FINDINGS: Data analysis revealed CCM specific proteins involved in endothelial dysfunction/inflammation/activation, leukocyte infiltration/chemotaxis, hemostasis, extracellular matrix dysfunction, astrocyte and microglial cell activation. Biomarker expression profiles matched bleeding status, especially with higher levels of inflammatory markers and activated astrocytes in ruptured than non-ruptured samples, some of these biomarkers are secreted into blood or urine. Furthermore, analysis was also done in a spatially resolving manner by separating the lesion area from the surrounding brain tissue. Our spatial studies revealed that although appearing histologically normal, the CCM border areas were pathological when compared to control brain tissues. Moreover, the functional relevance of CD93, ICAM-1 and MMP9, markers related to endothelial cell activation and extracellular matrix was validated by a murine pre-clinical CCM model. INTERPRETATION: Here we present a novel strategy for proteomics analysis on human CCMs, offering a possibility for high-throughput protein screening acquiring data on the local environment in the brain. Our data presented here describe CCM relevant brain proteins and specifically those which are secreted can serve the need of circulating CCM biomarkers to predict cavernoma's risk of bleeding.

C-C motif chemokine ligand 2 regulates prostaglandin synthesis and embryo attachment of the bovine endometrium during implantation.

C-C motif chemokine ligand 2 (CCL2) has been reported to be expressed in the bovine endometrium during pregnancy. However, the details of its functions involved in the implantation mechanism are still not clear. The purpose of this study is to analyze the functional properties of CCL2 in the bovine endometrium and embryos. The expression of CCR2 was not different between the luteal phase and implantation phase of their endometrial tissues, but was significantly high in IFNa treated bovine endometrial stromal (BES) cells in vitro. The expressions of PGES1, PGES2, AKR1C4, and AKR1C4 were high at the implantation stage compared with the luteal stage. On the other hand, PGES2 and AKR1B1 in BEE and PGES3 and AKR1A1 in BES were significantly increased by CCL2 treatment, respectively. The expressions of PCNA and IFNt were found significantly high in the bovine trophoblastic cells (BT) treated with CCL2 compared to the control. CCL2 significantly increased the attachment rate of BT vesicles to BEE in in vitro co-culture system. The expression of OPN and ICAM-1 increased in BEE, and ICAM-1 increased in BT by CCL2 treatment, respectively. The present results indicate that CCL2 has the potential to regulate the synthesis of PGs in the endometrium and the embryo growth. In addition, CCL2 has the possibility to regulate the process of bovine embryo attachment to the endometrium by modulation of binding molecules expression.

Amiodarone inhibits the Toll-like receptor 3-mediated nuclear factor κB signaling pathway by blocking organelle acidification.

Toll-like receptor (TLR) agonists or pro-inflammatory cytokines converge to activate the nuclear factor κB (NF-κB) signaling pathway, which provokes inflammatory responses. In the present study, we identified amiodarone hydrochloride as a selective inhibitor of the TLR3-mediated NF-κB signaling pathway by screening the RIKEN NPDepo Chemical Library. In human umbilical vein endothelial cells (HUVEC), amiodarone selectively inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) induced by polyinosinic-polycytidylic acid (Poly(I:C)), but not tumor necrosis factor-α, interleukin-1α, or lipopolysaccharide. In response to a Poly(I:C) stimulation, amiodarone at 20 µM reduced the up-regulation of mRNA expression encoding ICAM-1, vascular cell adhesion molecule-1, and E-selectin. The nuclear translocation of the NF-κB subunit RelA was inhibited by amiodarone at 15-20 µM in Poly(I:C)-stimulated HUVEC. Amiodarone diminished the fluorescent dots of LysoTracker® Red DND-99 scattered over the cytoplasm of HUVEC. Therefore, the present study revealed that amiodarone selectively inhibited the TLR3-mediated NF-κB signaling pathway by blocking the acidification of intracellular organelles.

Functional roles of CD26/DPP4 in lipopolysaccharide-induced lung injury.

Acute respiratory distress syndrome (ARDS) is characterized by dysregulated inflammation and increased permeability of lung microvascular cells. CD26/dipeptidyl peptidase-4 (DPP4) is a type II membrane protein that is expressed in several cell types and mediates multiple pleiotropic effects. We previously reported that DPP4 inhibition by sitagliptin attenuates lipopolysaccharide (LPS)-induced lung injury in mice. The current study characterized the functional role of CD26/DPP4 expression in LPS-induced lung injury in mice, isolated alveolar macrophages, and cultured lung endothelial cells. In LPS-induced lung injury, inflammatory responses [bronchoalveolar lavage fluid (BALF) neutrophil numbers and several proinflammatory cytokine levels] were attenuated in Dpp4 knockout (Dpp4 KO) mice. However, multiple assays of alveolar capillary permeability were similar between the Dpp4 KO and wild-type mice. TNF-α and IL-6 production was suppressed in alveolar macrophages isolated from Dpp4 KO mice. In contrast, in cultured mouse lung microvascular endothelial cells (MLMVECs), reduction in CD26/DPP4 expression by siRNA resulted in greater ICAM-1 and IL-6 expression after LPS stimulation. Moreover, the LPS-induced vascular monolayer permeability in vitro was higher in MLMVECs treated with Dpp4 siRNA, suggesting that CD26/DPP4 plays a protective role in endothelial barrier function. In summary, this study demonstrated that genetic deficiency of Dpp4 attenuates inflammatory responses but not permeability in LPS-induced lung injury in mice, potentially through differential functional roles of CD26/DPP4 expression in resident cellular components of the lung. CD26/DPP4 may be a potential therapeutic target for ARDS and warrants further exploration to precisely identify the multiple functional effects of CD26/DPP4 in ARDS pathophysiology.NEW & NOTEWORTHY We aimed to clarify the functional roles of CD26/DPP4 in ARDS pathophysiology using Dpp4-deficient mice and siRNA reduction techniques in cultured lung cells. Our results suggest that CD26/DPP4 expression plays a proinflammatory role in alveolar macrophages while also playing a protective role in the endothelial barrier. Dpp4 genetic deficiency attenuates inflammatory responses but not permeability in LPS-induced lung injury in mice, potentially through differential roles of CD26/DPP4 expression in the resident cellular components of the lung.

Advanced Glycation End Products Upregulate CD40 in Human Retinal Endothelial and Müller Cells: Relevance to Diabetic Retinopathy.

CD40 induces pro-inflammatory responses in endothelial and Müller cells and is required for the development of diabetic retinopathy (DR). CD40 is upregulated in these cells in patients with DR. CD40 upregulation is a central feature of CD40-driven inflammatory disorders. What drives CD40 upregulation in the diabetic retina remains unknown. We examined the role of advanced glycation end products (AGEs) in CD40 upregulation in endothelial cells and Müller cells. Human endothelial cells and Müller cells were incubated with unmodified or methylglyoxal (MGO)-modified fibronectin. CD40 expression was assessed by flow cytometry. The expression of ICAM-1 and CCL2 was examined by flow cytometry or ELISA after stimulation with CD154 (CD40 ligand). The expression of carboxymethyl lysine (CML), fibronectin, and laminin as well as CD40 in endothelial and Müller cells from patients with DR was examined by confocal microscopy. Fibronectin modified by MGO upregulated CD40 in endothelial and Müller cells. CD40 upregulation was functionally relevant. MGO-modified fibronectin enhanced CD154-driven upregulation of ICAM-1 and CCL2 in endothelial and Müller cells. Increased CD40 expression in endothelial and Müller cells from patients with DR was associated with increased CML expression in fibronectin and laminin. These findings identify AGEs as inducers of CD40 upregulation in endothelial and Müller cells and enhancers of CD40-dependent pro-inflammatory responses. CD40 upregulation in these cells is associated with higher CML expression in fibronectin and laminin in patients with DR. This study revealed that CD40 and AGEs, two important drivers of DR, are interconnected.

Targeting aberrant glycosylation to modulate microglial response and improve cognition in models of Alzheimer's disease.

Altered glycosylation profiles have been correlated with potential drug targets in various diseases, including Alzheimer's disease (AD). In this area, the linkage between bisecting N-acetylglucosamine (GlcNAc), a product of N-acetylglucosaminyltransferase III (GnT-III), and AD has been recognized, however, our understanding of the cause and the causative role of this aberrant glycosylation in AD are far from completion. Moreover, the effects and mechanisms of glycosylation-targeting interventions on memory and cognition, and novel targeting strategies are worth further study. Here, we showed the characteristic amyloid pathology-induced and age-related changes of GnT-III, and identified transcription factor 7-like 2 as the key transcription factor responsible for the abnormal expression of GnT-III in AD. Upregulation of GnT-III aggravated cognitive dysfunction and Alzheimer-like pathologies. In contrast, loss of GnT-III could improve cognition and alleviate pathologies. Furthermore, we found that an increase in bisecting GlcNAc modified ICAM-1 resulted in impairment of microglial responses, and genetic inactivation of GnT-III protected against AD mechanistically by blocking the aberrant glycosylation of ICAM-1 and subsequently modulating microglial responses, including microglial motility, phagocytosis ability, homeostatic/reactive state and neuroinflammation. Moreover, by target-based screening of GnT-III inhibitors from FDA-approved drug library, we identified two compounds, regorafenib and dihydroergocristine mesylate, showing pharmacological potential leading to modulation of aberrant glycosylation and microglial responses, and rescue of memory and cognition deficits.

Macrophage AHR-TLR4 cross-talk drives p-STAT3 (Ser727)-mediated mitochondrial oxidative stress and upregulates IDO/ICAM-1 in the steatohepatitis induced by aflatoxin B1.

Aflatoxin B1 (AFB1) is a major mycotoxin contaminant showing in the environment and foods. In this study, the molecular initiating events (MIEs) of AFB1-induced steatohepatitis were explored in mice and human cell model. We observed dose-dependent steatohepatitis in the AFB1-treated mice, including triglyceride accumulation, fibrotic collagen secretion, enrichment of CD11b + and F4/80+ macrophages/Kupffer cells, cell death, lymphocytes clusters and remarkable atrophy areas. The gut barrier and gut-microbiota were also severely damaged after the AFB1 treatment and pre-conditioned colitis in the experimental mice aggravated the steatohepatitis phenotypes. We found that macrophages cells can be pro-inflammatorily activated to M1-like phenotype by AFB1 through an AHR/TLR4/p-STAT3 (Ser727)-mediated mitochondrial oxidative stress. The phenotypes can be rescued by AHR inhibitors in the mice model and human cell model. We further showed that this signaling axis is based on the cross-talk interaction between AHR and TLR4. Gene knock-up experiment found that the signaling is dependent on AFB1 ligand-binding with AHR, but not protein expressions of TLR4. The signaling elevated NLRP3 and two immune metabolic enzymes ICAM-1 and IDO that are associated with macrophage polarization. Results from intervention experiments with natural anti-oxidant and AHR inhibitor CH223191 suggest that the macrophage polarization may rely on AHR and ROS. Our study provides novel and critical references to the food safety and public health regulation of AFB1.