Endothelial CD40 Research Articles

Reprogramming macrophage by targeting VEGF and CD40 potentiates OX40 immunotherapy

The low clinical response rate of checkpoint blockades, such as PD-1 and CTLA-4, highlighted the requirements of agonistic antibodies to boost optimal T cell responses. OX40, a co-stimulatory receptor on the T cells, plays a crucial role in promoting T cell survival and differentiation. However, the clinical efficacy of anti-OX40 agonistic antibodies was unimpressive. To explore the mechanism underlying the action of anti-OX40 agonists to improve the anti-tumor efficacy, we analyzed the dynamic changes of tumor-infiltrating immune cells at different days post-treatments using single-cell RNA-sequencing (scRNA-seq). In this study, we found that tumor-infiltrating regulatory T (Treg) cells were reduced after two rounds of anti-OX40 treatment, but the increase of infiltration and activation of CD8+ effector T cells, as well as M1 polarization in the tumor were only observed after three rounds of treatments. Moreover, our group first analyzed the antitumor effect of anti-OX40 treatments on regulating the macrophages and discovered the dynamic changes of vascular endothelial growth factor (VEGF) and CD40 signaling pathways on macrophages, indicating their possibility to being potential combination targets to improve the anti-OX40 agonists efficacy. The combination of VEGFR inhibitors or anti-CD40 agonist antibody with anti-OX40 agonists exhibited more remarkable inhibition of tumor growth. Therefore, the mechanism-driven combination of anti-OX40 agonists with VEGFR inhibitors or anti-CD40 agonists represented promising strategies.

Platelet factor 4 (PF4) induces cluster of differentiation 40 (CD40) expression in human aortic endothelial cells (HAECs) through the SIRT1/NF-κB/p65 signaling pathway.

PF4 is a pro-atherosclerotic molecule. Endothelial CD40, upon binding to its ligand CD40L, induces endothelial cell (EC) activation, which is a vital pathophysiological process in the initiation and progression of atherosclerosis. However, the relationship between PF4 and endothelial CD40 remains elusive. This study aims to investigate whether and how PF4 affects endothelial CD40 expression using primary HAECs. PF4 treatment down-regulated sirtuin 1 (SIRT1) expression but upregulated the expression of acetylated NF-κB p65 (Ac-p65) and CD40 in HAECs in a concentration- and time-dependent manner. Pretreatment with SIRT1 agonist (SRT1720 or RSV) or SIRT1-overexpressing lentivirus attenuated PF4-induced Ac-p65 and CD40 expression in HAECs, whereas preincubation with SIRT1 antagonist (NAM or EX527) or SIRT1 shRNA had the opposite effect. To investigate whether NF-κB/p65 signaling pathway modulates CD40 expression in PF4-treated HAECs, PDTC, a NF-κB inhibitor, and p65-shRNA were introduced. PDTC or p65-shRNA treatment down-regulated Ac-p65 expression in HAECs. PDTC or p65-shRNA preincubation suppressed CD40 expression in HAECs after PF4 treatment. To better determine whether SIRT1 regulates CD40 expression in PF4-treated HAECs via the NF-κB/p65 signaling pathway, p65-knockdown HAECs were preincubated with SIRT1 agonists before PF4 treatment. SIRT1 agonist preincubation further decreased CD40 expression in p65-knockdown HAECs treated with PF4. Moreover, PF4 treatment promoted p65 nuclear translocation in HAECs. The results of dual luciferase assay demonstrated that four NF-κB binding sites in the promoter of human CD40 gene were activated in PF4-treated HAECs. In conclusion, our findings suggest that PF4 treatment facilitates CD40 expression in HAECs through the SIRT1/NF-κB/p65 pathway.

Role of CD40 ligand-mediated endothelial cell-monocyte interaction at atherosclerosis predilection sites

BackgroundMonocyte recruitment into the vessel wall at atherosclerosis predilection sites is essential for lesion development in the early phase of atherosclerosis. Platelets interacting with ultra-large von Willebrand Factor (ULVWF) multimers deposited after CD40 receptor ligation on the endothelial surface form adhesive bridges and facilitate monocyte diapedesis. We hypothesise that enhanced endothelial CD40 expression at arterial bifurcations is responsible for monocyte recruitment and that its absence reduces susceptibility to atherosclerosis. MethodsY-shaped channel slides covered with endothelial cells (HUVEC) and isolated perfused carotid artery bifurcations from different mouse lines were used for adhesion studies with isolated fluorescent dye-labelled platelets and monocytes. Monocyte adherence was quantified via fluorescence imaging. Oil Red O staining visualised aortic atherosclerotic plaques, and mRNA expression was determined by qRT-PCR. ResultsIn response to soluble CD40 ligand (sCD40L) stimulated ULVWF release, the number of monocytes bound distal to the bifurcation of the Y-slide was 1.8-fold greater than without stimulation. The number of adherent monocytes in sCD40L-treated carotid artery bifurcations was 6 to 12.3-fold greater in ApoE knockout mice as compared to bifurcations derived from CD40/ApoE-deficient or control mice. CD40 mRNA expression was 2-fold higher in carotid artery bifurcations of ApoE knockout mice as compared to the proximal unbranched segment. Introduction of the CD40 knockout into the ApoE-/- background reduced the atherosclerosis burden along the entire aorta of these mice by 60 %. ConclusionsOur data demonstrate the importance of endothelial CD40 expression at atherosclerosis predilection sites for endothelial cell-platelet-monocyte interaction in the early phase of atherosclerosis.

CD40 Expressed in Endothelial Cells Promotes Upregulation of ICAM-1 But Not Pro-Inflammatory Cytokines, NOS2 and P2X7 in the Diabetic Retina.

PurposeCD40 is an upstream inducer of inflammation in the diabetic retina. CD40 is upregulated in retinal endothelial cells in diabetes. The purpose of this study was to determine whether expression of CD40 in endothelial cells is sufficient to promote inflammatory responses in the retina of diabetic mice.MethodsTransgenic mice with CD40 expression restricted to endothelial cells (Trg-CD40 EC), transgenic control mice (Trg-Ctr), B6, and CD40−/− mice were made diabetic using streptozotocin. Leukostasis was assessed using FITC-conjugated ConA. Pro-inflammatory molecule expression was examined by real-time PCR, immunohistochemistry, ELISA, or flow cytometry. Release of ATP was assessed by ATP bioluminescence.ResultsDiabetic B6 and Trg-CD40 EC mice exhibited increased retinal mRNA levels of ICAM-1, higher ICAM-1 expression in endothelial cells, and increased leukostasis. These responses were not detected in diabetic mice that lacked CD40 (CD40−/− and Trg-Ctr). Diabetic B6 but not Trg-CD40 EC mice upregulated TNF-α, IL-1β, and NOS2 mRNA levels. CD40 stimulation in retinal endothelial cells upregulated ICAM-1 but not TNF-α, IL-1β, or NOS2. CD40 ligation did not trigger ATP release by retinal endothelial cells or pro-inflammatory cytokine production in bystander myeloid cells. In contrast to diabetic B6 mice, diabetic Trg-CD40 EC mice did not upregulate P2X7 mRNA levels in the retina.ConclusionsEndothelial cell CD40 promotes ICAM-1 upregulation and leukostasis. In contrast, endothelial cell CD40 does not lead to pro-inflammatory cytokine and NOS2 upregulation likely because it does not activate purinergic-mediated pro-inflammatory molecule expression by myeloid cells or induce expression of these pro-inflammatory molecules in endothelial cells.

Deficiency of Endothelial CD40 Induces a Stable Plaque Phenotype and Limits Inflammatory Cell Recruitment to Atherosclerotic Lesions in Mice.

The co-stimulatory CD40L-CD40 dyad exerts a critical role in atherosclerosis by modulating leukocyte accumulation into developing atherosclerotic plaques. The requirement for cell-type specific expression of both molecules, however, remains elusive. Here, we evaluate the contribution of CD40 expressed on endothelial cells (ECs) in a mouse model of atherosclerosis. Atherosclerotic plaques of apolipoprotein E-deficient (Apoe -/- ) mice and humans displayed increased expression of CD40 on ECs compared with controls. To interrogate the role of CD40 on ECs in atherosclerosis, we induced EC-specific (BmxCreERT2-driven) deficiency of CD40 in Apoe -/- mice. After feeding a chow diet for 25 weeks, EC-specific deletion of CD40 (iEC-CD40) ameliorated plaque lipid deposition and lesional macrophage accumulation but increased intimal smooth muscle cell and collagen content, while atherosclerotic lesion size did not change. Leukocyte adhesion to the vessel wall was impaired in iEC-CD40-deficient mice as demonstrated by intravital microscopy. In accord, expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) in the vascular endothelium declined after deletion of CD40. In vitro, antibody-mediated inhibition of human endothelial CD40 significantly abated monocyte adhesion on ECs. Endothelial deficiency of CD40 in mice promotes structural features associated with a stable plaque phenotype in humans and decreases leukocyte adhesion. These results suggest that endothelial-expressed CD40 contributes to inflammatory cell migration and consecutive plaque formation in atherogenesis.

Tumor necrosis factor receptor type 1-associated death domain (TRADD) regulates epithelial-mesenchymal transition (EMT), M1/M2 macrophage polarization and ectopic endometrial cysts formation in endometriosis.

BackgroundEndometriosis is a gynecological non-malignant disease that is manifested by the presence of extrauterine ectopic endometrial cells and stroma. The aim of current study was to explore the role of tumor necrosis factor receptor type 1-associated death domain (TRADD) protein in endometriosis.MethodsCell migration, invasion, and the expression of epithelial-mesenchymal transition (EMT) inducers in TRADD silencing or overexpression in eutopic endometrial stromal cells (EuSCs) and ectopic endometrial stromal cells (EcSCs) were analyzed by wound healing assay, transwell assay, quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting and rat endometriosis model. A cell line derived from THP-1 macrophages was used to measure M1/M2 polarization in endometriosis by flow cytometry and enzyme-linked immunosorbent assay (ELISA).ResultsThe mRNA level and protein expression of TRADD, keratin, E-cadherin, N-cadherin, vascular endothelial growth factor, matrix metalloproteinase-9, CD40, and CD206 were abnormally expressed in ectopic endometrial tissues and EcSCs. TRADD silencing promoted migration, invasion, and EMT in EuSCs, while TRADD overexpression restrained migration, invasion, and EMT in EcSCs. TRADD knockdown prohibited M1/M2 polarization in normal endometrial homogenization-treated THP-1-derived macrophages, whereas TRADD upregulation facilitated M1/M2 polarization in patients with endometrial homogenization-treated THP-1-derived macrophages. In addition, TRADD overexpression suppressed ectopic endometrial cysts formation in a rat endometriosis model. TRADD overexpression activated NF-κB and MAPK signaling in EcSCs and rat models.ConclusionsOur results indicated that the overexpression of TRADD prohibited migration, invasion, EMT, M1/M2 polarization and ectopic endometrial cysts formation in endometriosis, and this might due to regulating NF-κB and MAPK signaling.

CD40 in Endothelial Cells Restricts Neural Tissue Invasion by Toxoplasma gondii.

Little is known about whether pathogen invasion of neural tissue is affected by immune-based mechanisms in endothelial cells. We examined the effects of endothelial cell CD40 on Toxoplasma gondii invasion of the retina and brain, organs seeded hematogenously. T. gondii circulates in the bloodstream within infected leukocytes (including monocytes and dendritic cells) and as extracellular tachyzoites. After T. gondii infection, mice that expressed CD40 restricted to endothelial cells exhibited diminished parasite loads and histopathology in the retina and brain. These mice also had lower parasite loads in the retina and brain after intravenous (i.v.) injection of infected monocytes or dendritic cells. The protective effect of endothelial cell CD40 was not explained by changes in cellular or humoral immunity, reduced transmigration of leukocytes into neural tissue, or reduced invasion by extracellular parasites. Circulating T. gondii-infected leukocytes (dendritic cells used as a model) led to infection of neural endothelial cells. The number of foci of infection in these cells were reduced if endothelial cells expressed CD40. Infected dendritic cells and macrophages expressed membrane-associated inducible Hsp70. Infected leukocytes triggered Hsp70-dependent autophagy in CD40+ endothelial cells and anti-T. gondii activity dependent on ULK1 and beclin 1. Reduced parasite load in the retina and brain not only required CD40 expression in endothelial cells but was also dependent on beclin 1 and the expression of inducible Hsp70 in dendritic cells. These studies suggest that during endothelial cell-leukocyte interaction, CD40 restricts T. gondii invasion of neural tissue through a mechanism that appears mediated by endothelial cell anti-parasitic activity stimulated by Hsp70.

Up-regulation of fibrinogen-like protein 2 in porcine endothelial cells by xenogeneic CD40 signal

ABSTRACTAcute humoral xenograft rejection (AHXR), characterized by thrombin generation and endothelial cell activation, should be overcome for the success of xenotransplantation. Fibrinogen-like protein 2 (fgl2) expressed on endothelial cells can convert prothrombin to thrombin directly, which indicates that the induced fgl2 expression in activated endothelial cells can contribute to thrombosis. In xenotransplant condition, the interaction between human CD40L and porcine endothelial CD40 can activate endothelial cells. In this study, we investigated the effect of endothelial cell activation through the interaction between human CD40L and porcine CD40 on fgl2 expression and its function as a direct prothrombinase. We found that CD40 stimulation up-regulated fgl2 expression as well as its enzymatic activity in porcine endothelial cells. Moreover, functional studies using knock-down system showed that the major factor converting human prothrombin to thrombin is fgl2 protein expressed on porcine endothelial cells. Overall, this study demonstrates that fgl2 expression can be induced by xenogeneic CD40 signal on endothelial cells and contribute to thrombin generation.

A PPAR\u03b3-dependent miR-424/503-CD40 axis regulates inflammation mediated angiogenesis

Activation of the endothelium by pro-inflammatory stimuli plays a key role in the pathogenesis of a multitude of vascular diseases. Angiogenesis is a crucial component of the vascular response associated with inflammatory signaling. The CD40/CD40 ligand dyad in endothelial cells (EC) has a central role in promoting vascular inflammatory response; however, the molecular mechanism underlying this component of inflammation and angiogenesis is not fully understood. Here we report a novel microRNA mediated suppression of endothelial CD40 expression. We found that CD40 is closely regulated by miR-424 and miR-503, which directly target its 3′ untranslated region. Pro-inflammatory stimuli led to increased endothelial CD40 expression, at least in part due to decreased miR-424 and miR-503 expression. In addition, miR-424 and miR-503 reduced LPS induced EC sprouting, migration and tube formation. Moreover, we found that miR-424 and miR-503 expression is directly regulated by peroxisome proliferator-activated receptor gamma (PPARγ), whose endothelial expression and activity are decreased in response to inflammatory factors. Finally, we demonstrate that mice with endothelial-specific deletion of miR-322 (miR-424 ortholog) and miR-503 have augmented angiogenic response to LPS in a Matrigel plug assay. Overall, these studies identify a PPARγ-dependent miR-424/503-CD40 signaling axis that is critical for regulation of inflammation mediated angiogenesis.

H2S dependent and independent anti-inflammatory activity of zofenoprilat in cells of the vascular wall

Cardiovascular diseases as atherosclerosis are associated to an inflammatory state of the vessel wall which is accompanied by endothelial dysfunction, and adherence and activation of circulating inflammatory cells. Hydrogen sulfide, a novel cardiovascular protective gaseous mediator, has been reported to exert anti-inflammatory activity. We have recently demonstrated that the SH containing ACE inhibitor zofenoprilat, the active metabolite of zofenopril, controls the angiogenic features of vascular endothelium through H2S enzymatic production by cystathionine gamma lyase (CSE). Based on H2S donor/generator property of zofenoprilat, the objective of this study was to evaluate whether zofenoprilat exerts anti-inflammatory activity in vascular cells through its ability to increase H2S availability.Here we found that zofenoprilat, in a CSE/H2S-mediated manner, abolished all the inflammatory features induced by interlukin-1beta (IL-1β) in human umbilical vein endothelial cells (HUVEC), especially the NF-κB/cyclooxygenase-2 (COX-2)/prostanoid biochemical pathway. The pre-incubation with zofenoprilat/CSE dependent H2S prevented IL-1β induced paracellular hyperpermeability through the control of expression and localization of cell-cell junctional markers ZO-1 and VE-cadherin. Moreover, zofenoprilat/CSE dependent H2S reduced the expression of the endothelial markers CD40 and CD31, involved in the recruitment of circulating mononuclear cells and platelets. Interestingly, this anti-inflammatory activity was also confirmed in vascular smooth muscle cells and fibroblasts as zofenoprilat reduced, in both cell lines, proliferation, migration and COX-2 expression induced by IL-1β, but independently from the SH moiety and H2S availability.These in vitro data document the anti-inflammatory activity of zofenoprilat on vascular cells, reinforcing the cardiovascular protective effect of this multitasking drug.

Phloretin suppresses thrombin‐mediated leukocyte‐platelet‐endothelial interactions

Thrombin playing a pivotal role in coagulation cascade may influence the onset and progression of atherosclerosis as a pro-inflammatory mediator. This study investigated whether phloretin found in apple tree leaves, severed a linkage between thrombosis and atherosclerosis by thrombin. Human endothelial cells were pre-treated with 1-20 μM phloretin and stimulated with 10 U/mL thrombin. Phloretin attenuated adhesion of THP-1 monocytes and platelets to thrombin-inflamed endothelial cells with concurrent inhibition of protease-activated receptor (PAR-1) induction. The thrombin induction of endothelial CD40, endothelial integrin β3 and P-selectin, and monocytic CD40L was dampened by phloretin. Additionally, phloretin inhibited monocyte secretion of MCP-1, IL-6 and IL-8 responsible for pro-inflammatory activity of thrombin inducing endothelial CD40. The monocyte COX-2 induction and PGE2 secretion due to thrombin were down-regulated by phloretin, deterring endothelial CD40 expression. Thrombin promoted production of PAI-1 and tissue factor in monocytes was attenuated by phloretin through blocking PAR-1 and CD40. Thrombin up-regulated the induction of endothelial connective tissue growth factor independent of PAR-1 activation, which was reversed by phloretin. Phloretin disturbed tethering and stable adhesion of monocytes and platelets onto endothelium during increased thrombosis by thrombin. Phloretin would be a potent agent preventing thrombosis and atherosclerosis.

Lack of evidence of CD40 ligand involvement in transfusion-related acute lung injury

Activated platelets have been implicated in playing a major role in transfusion-related acute lung injury (TRALI), as platelets can trigger neutrophils, resulting in vascular damage. We hypothesized that binding of platelet CD40 ligand (CD40L) to endothelial CD40 is essential in the onset of TRALI. Mice were challenged with monoclonal major histocompatibility complex (MHC)-1 antibody which induced TRALI, evidenced by pulmonary oedema, accompanied by significantly elevated bronchoalveolar fluid (BALF) levels of total protein and elevated plasma levels of keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2) compared to infusion of isotype antibody (all Ps < 0·05). Treatment with ciglitazone, which inhibits platelet CD40L expression, had no effect on pulmonary and systemic inflammation compared to controls. In addition, treatment with anti-CD40L antibody, which antagonizes all CD40-CD40L interactions, also did not abrogate the TRALI reaction. Furthermore, levels of soluble CD40L were measured in a cohort of cardiac surgery patients, who were followed prospectively for the onset of TRALI after transfusion. Plasma levels of sCD40L at baseline and at time of developing TRALI did not differ between TRALI patients and controls (transfused cardiac surgery patients not developing acute lung injury) (275 ± 192 versus 258 ± 346 and 93 ± 82 versus 93 ± 123 pg/ml, respectively, not significant). In conclusion, these results do not support the idea that the CD40-CD40L interaction is involved in mediating TRALI.

Interaction with damaged vessel wall in vivo in humans induces platelets to express CD40L resulting in endothelial activation with no effect of aspirin intake

Activated platelets express CD40L on their plasma membrane and release the soluble fragment sCD40L. The interaction between platelet surface CD40L and endothelial cell CD40 leads to the activation of endothelium contributing to atherothrombosis. Few studies have directly demonstrated an increased expression of platelet CD40L in conditions of in vivo platelet activation in humans, and no data are available on its relevance for endothelial activation. We aimed to assess whether platelets activated in vivo at a localized site of vascular injury in humans express CD40L and release sCD40L, whether the level of platelet CD40L expression attained in vivo is sufficient to induce endothelial activation, and whether platelet CD40L expression is inhibited by aspirin intake. We used the skin-bleeding-time test as a model to study the interaction between platelets and a damaged vessel wall by measuring CD40L in the blood emerging from a skin wound in vivo in healthy volunteers. In some experiments, shed blood was analyzed before and 1 h after the intake of 500 mg of aspirin. Platelets from the bleeding-time blood express CD40L and release soluble sCD40L, in a time-dependent way. In vivo platelet CD40L expression was mild but sufficient to induce VCAM-1 expression and IL-8 secretion in coincubation experiments with cultured human endothelial cells. Moreover, platelets recovered from the bleeding-time blood activated endothelial cells; an anti-CD40L antibody blocked this effect. On the contrary, the amount of sCD40L released by activated platelets at a localized site of vascular injury did not reach the concentrations required to induce endothelial cell activation. Soluble monocyte chemoattractant protein-1, a marker of endothelium activation, was increased in shed blood and correlated with platelet CD40L expression. Aspirin intake did not inhibit CD40L expression by platelets in vivo. We concluded that CD40L expressed by platelets in vivo in humans upon contact with a damaged vessel wall activates endothelium; aspirin treatment does not inhibit this mechanism.

Effect of Enhanced Glycemic Control with Saxagliptin on Endothelial Nitric Oxide Release and CD40 Levels in Obese Rats

Endothelial cell (EC) dysfunction contributes to insulin resistance in diabetes and is characterized by reduced nitric oxide (NO) release, increased nitroxidative stress and enhanced inflammation. The purpose of this study was to test the effect of improved postprandial glucose control on EC function in insulin-resistant rats as compared to fasting glucose (FG) changes. Obese Zucker rats were treated with 10 mg/kg/day saxagliptin, a dipeptidyl peptidase-4 (DPP4) inhibitor, for 4 or 8 weeks and compared to lean rats. NO and peroxynitrite (ONOO(-)) release from aortic and glomerular ECs was measured ex vivo using amperometric approaches and correlated with FG, postprandial glucose, insulin, soluble CD40 (sCD40) and L-citrulline levels. Saxagliptin treatment improved NO production and reduced ONOO(-) release prior to any observed changes in FG levels. In untreated obese animals, NO release from aortic and glomerular ECs decreased by 22% and 31%, respectively, while ONOO(-) release increased by 26% and 40%. Saxagliptin increased aortic and glomerular NO release by 18% and 31%, respectively, with comparable reductions in ONOO(-) levels; the NO/ONOO(-) ratio, an indicator of NO synthase coupling, increased by >40%. Improved glycemic control was further associated with a reduction in sCD40 levels by more than ten-fold (from 300 ± 206 to 22 ± 22 pg/mL, p < 0.001). These findings indicate that enhanced glycemic control with DPP4 inhibition improved NO release and reduced inflammation in a manner not predicted by FG changes alone.

CD40/CD40L interaction induces E-selectin dependent leukocyte adhesion to human endothelial cells and inhibits endothelial cell migration

BackgroundCD40 is a receptor expressed on a wide range of cells such as leukocytes and endothelial cells (EC). As a member of the tumor necrosis factor (TNF) superfamily the activation of CD40 by CD40-ligand (CD40L) plays a crucial role for the development and progression of a variety of inflammatory processes including atherosclerosis. The aim of the present study was to investigate the effect of CD40/CD40L interaction on leukocyte adhesion to the endothelium and on endothelial cell migration. Methods and resultsHuman umbilical vein endothelial cells (HUVEC) were stimulated with either stable transfectants of mouse myeloma cells expressing the CD40L or wild type cells (4h). Subsequently adhesion of leukocytes expressing Sialyl Lewis X, the counterpart for E-selectin (HL60 cells), was measured under shear stress (2–2.6dyne/cm2) using a flow chamber adhesion assay. Stimulation of CD40 led to a significant increase of E-selectin dependent adhesion of leukocytes to the endothelium. Incubation of cells with either the CD40L blocking antibody TRAP-1 or the E-selectin blocking antibody BBA2 during CD40 stimulation completely abolished adhesion of leukocytes to HUVEC. Similar results were found in human cardiac microvasculature endothelial cells (HCMEC). In contrast stimulation of CD40 had no effect on adhesion of l-selectin expressing NALM6-L cells. Furthermore, CD40/CD40L interaction abrogated VEGF-induced migration of HUVEC compared to non-stimulated controls. In comparison experiments, stimulation of endothelial cells with VEGF led to a significant phosphorylation of ERK1/2, Akt, and eNOS. Stimulation of endothelial CD40 had no effect on VEGF-induced phosphorylation of ERK1/2. However, VEGF-induced activation of Akt and eNOS was reduced to baseline levels when endothelial CD40 was stimulated. ConclusionCD40/CD40L interaction induces E-selectin dependent adhesion of leukocytes to human endothelial cells and reduces endothelial cell migration by inhibiting the Akt/eNOS signaling pathway.

Deficiencies in the CD40 and CD154 receptor-ligand system reduce experimental lung metastasis

It is established that experimental metastasis requires platelet activity. CD154 expressed on and released from activated platelets induces an inflammatory response in endothelial cells and monocytes, including tissue factor production. CD154 has also been shown to activate platelets in vitro and promote thrombus stability in vivo. These CD154 effects may be mediated, at least in part, by CD40 signaling on platelets and vascular endothelial cells. We have previously demonstrated prolonged bleeding and PFA-100 closure times in mice deficient for Cd154 or its receptor Cd40. In the present study, we hypothesized that Cd40 and Cd154 promote lung tumor formation in experimental metastasis in mice. We created mice doubly deficient in Cd40 and Cd154 (Dbl KO) and found them to be both fertile and viable. Injected tumor cells seeded poorly in mice deficient in Cd40 or Cd154, as well as Dbl KO, compared to wild-type mice. We sought to determine whether blood-borne Cd40 versus endothelial Cd40 contribute differentially to reduced experimental lung metastasis, as observed in Cd40 deficient mice. By bone marrow transplantation, we created mice deficient for Cd40 either in the blood compartment but not in the endothelium, or vice versa. We found that mice deficient in blood compartment Cd40 had fewer lung nodules compared to wild-type mice and mice deficient in endothelial Cd40. Our findings suggest an important contribution of the Cd40-Cd154 pathway to experimental lung metastasis. Furthermore, the data points to a selective role for peripheral blood cell Cd40 in this process.

CD40 activation is angioproliferative in pulmonary microvascular endothelial cells

IntroductionEndothelial CD40 is important during inflammation as an angiogenic stimulus. We tested whether CD40 expression in PMVEC is associated with angioproliferative potential.MethodsWestern analysis for CD40 expression was performed. Growth curves, barrier measurements, and scratch‐wound assays were carried out using an ECIS apparatus (Applied Biophysics) and a digital image system composed of an inverted microscope.ResultsBasal CD40 expression was high in PMVEC, whereas expression was absent in rat pulmonary arterial‐derived EC (PAEC). CD40 expression could be up‐regulated in PAEC following over‐expression of NAP‐1, an endothelial progenitor cell‐associated protein. Exposure of PMVEC to sCD40L (1 μg/mL) for 1 ‐ 48 hours increased proliferation and improved barrier integrity. Exposure to sCD40L also produced an accelerated PMVEC migration response with decreased wound closure times. These effects were inhibited by pre‐treatment with anti‐CD40 antibodies.ConclusionsOur data indicate activation of PMVEC‐expressed CD40 results in angioproliferative responses. These responses may be inherent to cell phenotypes expressing NAP‐1. Identification of PMVEC expressing CD40 in situ may localize pulmonary resident endothelial progenitor cells. Support: Parker B. Francis Foundation and AHA (0765421B) to T.M.M.

CD40: an upstream master switch for endothelial cell activation uncovered by RNAi-coupled transcriptional profiling

The CD40-CD154 dyad seems to play a prominent role fostering the immune-inflammatory response triggered by endothelial cell (EC)-T-cell communication. To delineate comprehensively the involvement of CD40 (TNFRSF5) in EC activation, we combined RNAi-mediated CD40 knockdown with comparative genome-wide transcriptional profiling of ECs interacting with (CD154+) T cells. We report the initiation of a profound stress response in ECs upon CD40-CD154 engagement through early up-regulation of, among others, the major proinflammatory NF-kappaB and MAPK/SAPK pathways and their associated transcription factors. Moreover, we have identified novel genes regulated through the CD40-CD154 interaction, and pathways previously unrecognized to be induced by CD40 signaling in ECs. Thus, we document a significant down-regulation of endothelial APLN by CD40-CD154 interaction, TNFalpha/IFNgamma exposure, and in immune-inflammatory pathologies, which could lead to hemodynamic dysfunction. Conversely, CD40-mediated up-regulation of the viral immune surveillance system, notably TLR3, IFIH1, RIG-I, and RNASEL, establishes a reverse link from adaptive to innate immunity in ECs. Moreover, systematic enrichment analysis substantiates endothelial CD40 involvement in the transcriptional regulation of gene networks associated with adhesion and motility, immunity, cell fate control, hemostasis, and metabolism. Our study also highlights the anti-inflammatory potential of RNAi-mediated CD40 inhibition, and the relevance of CD40 signaling for therapeutic intervention.

Xenogeneic interaction between human CD40L and porcine CD40 activates porcine endothelial cells through NF-κB signaling

Xenotransplantation is a promising alternative to overcome donor shortage in transplantation. CD40 molecule plays an important role in the interaction of T cells with antigen-presenting cells and in the activation of vascular endothelial cells. We investigated whether the xenogeneic interaction between human CD40L (hCD40L) on T cells and porcine endothelial CD40 (pCD40) can activate porcine endothelial cells (PECs). The interaction between hCD40L and pCD40 induced the expression of chemokines on PECs as well as MHC and adhesion molecules. Furthermore, NF-κB signaling was activated in HEK 293 cells expressing pCD40 and PECs by stimulation with hCD40L+ Jurkat T clones. Both anti-CD40L neutralizing antibodies and NF-κB signal inhibitors interfered with immune activation of PECs. Overall, this study shows that xenogeneic interaction between hCD40L and pCD40 can activate PECs through NF-κB signaling, and therefore may contribute to acute vascular rejection in xenotransplantation.

STAT-1 decoy oligonucleotide improves microcirculation and reduces acute rejection in allogeneic rat small bowel transplants

During acute rejection leukocyte-endothelial cell interaction fuelled by costimulatory molecules such as the CD40/CD154 receptor/ligand dyad disrupts microcirculation of the small bowel. Downregulating endothelial CD40 expression by employing a decoy oligonucleotide (dODN) neutralizing the transcription factor signal transducer and activator of transcription-1 (STAT-1) may protect the graft. Therefore allogenic small bowel transplantation was performed in the Brown Norway to Lewis rat model. Graft vessels were pretreated with STAT-1 dODN, mutant control ODN (20 microM) or vehicle (n=8). CD40 antisense ODN and scrambled control ODN-treated transplants served as target control (n=3 each). Intravital microscopy, histology, immunohistochemistry and Western blot analyses were performed 7 days later. Functional capillary density, red blood cell velocity and perfusion index in STAT-1 dODN and CD40 antisense ODN-treated transplants were improved whereas stasis index was reduced. Leukocyte-endothelial cell interaction showed no difference. Histological parameters of rejection, infiltrating CD3-positive cells and apoptotic bodies were also reduced in STAT-1 dODN and CD40 antisense ODN-treated transplants 7 days post-transplantation. CD40 protein abundance was reduced to less than 10% of control in STAT-1 dODN-treated grafts. STAT-1 dODN blockade of CD40 expression improves mucosal perfusion, reduces graft rejection, T-cell infiltration and apoptosis in rat small bowel allografts during acute rejection.