Activation Of OX40L Reverse Signal Pathway Potentiates Intracellular Free Calcium Concentration In Cultured Aortic Endothelial Cells From C57BL/6J Mice

BackgroundCo-stimulatory molecules OX40/OX40L play an important role in mediation signal transduction in the development and process of atherosclerosis. OX40 ligand (OX40L/CD134L/gp34), an accessory molecule of immune response, belongs to the TNF super-family. OX40L is primarily found on activated antigen presenting cell, including dendritic cells,B-lymphocyte,macrophages,vascular endothelial cells,heart,skeletal muscle and lung. The receptor of OX40L, OX40, is primarily induced on activated T cells. OX40/OX40L mediated co-stimulation signal maintains T cells survival; promotes T cells proliferation and generation of memory T cells; enhances adhesion between T-cells and other cells; transmits stimulant information of antigens; participates process of cells immune activation. Interruption of OX40/OX40L interaction has been shown to ameliorate inflammatory reaction in the inflamed site instead of global inflammatory reaction. Hence, it's an excellent target for inflammatory-mediated diseases therapy.Up to 2005, Jackson Laboratory firstly reported that OX40/OX40L participate in atherosclerotic disease process. Anti-OX40L antibody treatment leaded to a marked decrease of atherosclerotic plaque and promoted plaque stabilization. The expression of OX40L was found in all the major cell types in atherosclerotic lesions, including endothelial cells, smooth muscle cell, macrophages, T-lymphocytes. OX40L is expressed more highly in plaque tissue than in normal. Apart from the role to deliver costimulatory signals, are there other biological effects exist? What's the role in atherosclerotic plaque rupture? OX40/OX40L, a pair of co-stimulatory molecules, generates bi-directional signals and may elicit respective biological effects. It is an important characteristic that ECs OX40L directly mediates T-cells or OX40+ T cells adhesion to ECs and T-lymphocytes gather at atherosclerosis plaques. Enhanced expression of ECs OX40L in plaque and activation of OX40L reverse signal potentiates signal transduction between T cells and ECs, which could lead to the disequilibrium of expression and secretion between plaque stable factor and plaque unstable factor.Elevated cytosolic free Ca²⁺ is a trigger in many signal transduction, which could lead to the secretion of a variety of endothelium-derived atherogenic factors and vasoconstrictor. For instance, endothelin,von willebrand factor,tissue factor and prostaglandin E2. These factors have been confirmed harmful, causing inflammation, increasing plaque instability. OX40, a known proinflammatory factors, maybe involved in [Ca²⁺]_i overproduction. Hence, we investigated the changes of [Ca²⁺]_i in MAECs subsequently.ObjectThe present study was designed to investigate the effect of intracellular free calcium concentration of aortic endothelial Cells of C57BL/6J mice after activation of OX40L reverse signal pathway.Materials and methods1. C57BL/6J Mice, aged 8 to 12 weeks, was treated with an intraperitoneal injection of heparin sodium (1250U), and then anesthetized with 11.25g/kg chloral hydrate. The midline of the abdomen is incised. Then the thorax was opened to expose the thoracic aorta and abdominal aorta. The abdiminal aorta is cut at the site of renal artery to release the blood, and then perfused with 1 ml phosphate buffered saline containing 1000U/ml of heparin from the apex of left ventricle. The thoracic aorta was cannulated by a 23-gaug cannula. Then the thoracic aorta was filled with collagenase type II solution to dissociate for 45 min at 37°C. Collagenase action was stopped and detached cells were obtained by perfusing the thoracic aorta with DMEM supplemented with 20% fetal calf serum and 1% penicillin-streptomycin. The perfusate was centrifuged at 1000rpm for 5 min and the pellet was resuspended with endothelial cell culture media and maintained in the same solution and cultured in 6 well-plate coated with collagen type I in a humidified atmosphere of 5% CO₂ at 37°C. The medium was removed after 2 h and covered with fresh culture medium for 3 days. Fresh endothelial cells culture medium was changed every 2 to 3 days.2. Confluent monolayers were fixed with chilled methanol, and endogenous peroxidases were quenched by 0.06% H₂O₂. Cells were subsequently blocked with goat serum used to generate the secondary antibody. ECs were characterization using anti-vWF antibody (1:400 dilution) and smooth muscle cells were detected by an antibody against alpha actin(1:150 dilution). Primary antisera were incubated at 4°C overnight, followed by an incubation of a secondary horseradish peroxidase (HRP) conjugated antibody (1:200dilution) under 37°C for 1h. Immunoreactivity was visualized by incubation with the DAB kit.3. Confluent monolayers were fixed with chilled methanol for 20min at -20°C, Endogenous peroxidases were blocked by 15min incubation in 0.06% H₂O₂. Samples were subsequently blocked with serum used to generate the secondary antibody. We then applied an antibody against mouse OX40L (1:200dilution). The specific binding was detected with HRP labeled secondary antibody (1:200 dilution) and DAB as a substrate. The cells staining images were produced in an OLYMPUS inverted microscope equipped with a Cannon digital camera and underwent adjustment of brightness and contrast using Adobe Photoshop 7.0.4. ECs were assigned to these groups: 1) ECs, not receiving any treatment, was performed as control. 2) ECs, subjected to indicated OX40 concentrations stimulation (0, 25, 50, 75, 100, 125 ug/L) at 37°C for 30min, were used to investigate the dose-dependent manner of OX40-induced [Ca²⁺]_i ;3) ECs, pretreated with the selective and competive OX40 inhibitor anti-OX40L antibody (100ug/L) prior to adding OX40(0, 25, 50, 75, 100ug/L), were used to investigate the down-regulation of OX40 stimulation. 4) ECs, subjected to indicated stimulation time (24h, 48h,72h) at 37°C, were performed to investigate the time manner of OX40-induced[Ca²⁺]_i. Then the cells were loaded with 4umol/L calcium sensitive probe Fura-2/AM for 30 min at 37°C in the dark. Under inverted microscope: the excitation wavelengths were 340 and 380 nm, and the emitted fluorescence was measured at 510 nm. Intracellular free calcium was analyzed as the ratio of background-corrected fluorescence at 340 nm excitation to 380 nm excitation (F340/F380 ratio). Changes in the ratio of F340/F380 were regarded as changes in [Ca²⁺]_i.Results1. Fresh cells effluents contained small clumps is rounded droplet, which spread to form small epithelioid clusters within the first 3d in culture. These clusters increased in size and gradually coalesced to form incomplete mono-layers by 7-10 days. Cells within these early mono-layers were uniform in appearance: elongated, with single ovoid nuclei containing one to two prominent nucleoli, and a broad, thin peripheral cytoplasm with distinct borders. Then, single cell-thick confluent mono-layers of densely packed polygonal cells had formed, presenting with a cobblestone appearance, a characteristic morphology of ECs. Immunocytochemical characterization of cells showed a positive staining for vWF whereas alpha-actin staining and negative control staining was negative.2. OX40L was expressed on cultured vascular endothelial cells by ECs OX40L immunocytochemical staining. Under inverted microscope, all ECs were OX40L positive staining cells in 5 random visual fields.3. Effects of OX40L reverse signal pathway on intracellular calcium concentration in aortic endothelial Cells of C57BL/6J mice:3.1 When cells were subjected to 25ug/L OX40 stimulation at 37°C for 30min, there was no change in [Ca²⁺]_i of ECs (P>0.05). The OX40 increased [Ca²⁺]_i was observed at 50ug/L and significantly increased in dose-dependent manner from 50ug/L to 125ug/L ((P<0.05)). And there was a peak at 100ug/L OX40 stimulation. However, there was no difference in [Ca²⁺]_i of ECs between 100ug/L and 125ug/L(P>0.05).3.2 There was no difference in[Ca²⁺]_i before or after adding anti-OX40L antibody(P>0.05). However, in the selective OX40 inhibitor anti-OX40L antibody pretreated cells (100ugl/L for 30min) prior to adding OX40 (50,75 , 100ug/L), this increase of[Ca²⁺]_i was significantly abrogated(P<0.05).3.3 When cells were subjected to 100ug/L OX40 stimulation at 37°C for different times (24h, 48h, 72h), there was no change in [Ca²⁺]_i of ECs at 24h(P>0.05). [Ca²⁺]_i of EC significantly increased after the treatment of OX40 in 48h and 72h(P<0.05).Conclusions:1. The present study established a method to isolate and culture aortic endothelial cells from mice which was confirmed by endothelial cell morphology and immunocytochemical staining.2. The cultured aortic endothelial cells from C57BL/6J mice expressed OX40L.3. The results indicated that the activation of OX40L reverse signal can increase the intracellular free calcium concentration of mouse aortic endothelial cells. The elevated of intracellular free calcium can partly explain the effect of OX/OX40L on the pathyology of vascular disorder.