Inhibitory Effect And Molecular Mechanisms Of Genipin On Exocytosis In Human Umbilical Vein Endothelial Cells

Endothelail cells lining in inner surface of vasculature provide a physical barrier between circulating blood and vessel wall. It is not only a barrier in biological structure and function, but also an active metabolism pool which synthesizes numerous bioactive product. Endothelial cell dyfunction and injury which play important roles in cardio-cerebrovascular disease are tightly associated with initial stage of various diseases. In the development of endothelial cell dyfunction and injury, exocytosis of endothelial cell grannules is one of the earliest responses to injury or stimmulation of vascular issue. When endothelial cells are stimulated, the membranes of Weibel-Palade bodies (WPB) rapidly fuse with the endothelial plasma membrane, releasing their contents into outside of the endothelial cells. The proteins released from WPB exocytosis promote neutrophils and platelet adhesion to vessel walls, and cause vascular inflammation and thrombogensis. Therefore, exocytosis becomes an important aspect for exploring the mechanism of vascualr dysfunction and injury.WPB mainly stores in endothelial cells and contains pro-inflammatory as well as pro-thrombotic proteins such as P-selectin, E-selectin, vascular endothelial cell adhesion molecule-1(VCAM-1) and von Willebrand factor (VWF). Most of them are related with cell adhesion, inflammation and hemostasis. P-selectin is a cell adhesion molecule (CAM) on the surfaces of activated endothelial cells. It plays an essential role in the initial recruitment of leukocytes to the site of injury in the early stage of inflammation. In the advanced stage, P-selectin mediates adhesion event with other adhesion molecules. P-selectin is also responsible for the rolling phase of the leukocyte adhesion cascade. When endothelial cells are activated by molecules such as thrombin and histamine, P-selectin moves from an internal cell location to the endothelial cell surface. E-selectin, also known as CD62e, is a cell adhesion molecule expressed only on endothelial cells. E-selectin recognizes and binds to sialylated carbohydrates present on the surface proteins of certain leukocytes. These carbohydrates include members of the Lewis X and Lewis. During inflammation, E-selectin recruits leukocytes to the site of injury. The local release of cytokines IL-1 and TNF by damaged cells induces the over-expression of E-selectin on endothelial cells of nearby blood vessels. Leukocytes in the blood expressing the correct ligand will bind with low affinity to E-selectin, causing the leukocytes to roll along the internal surface of the blood vessel. As the inflammatory response progresses, chemokines released by injured tissue enter the blood vessels and activate the rolling leukocytes, which are now able to tightly bind to the endothelial surface and begin making their way into the tissue. The VCAM-1 protein is an endothelial ligand for VLA-4 (Very Late Antigen-4 or a4β1) of theβ1 subfamily of integrins, and for integrin a4β7. The VCAM-1 protein mediates the adhesion of lymphocytes, monocytes, eosinophils, and basophils to vascular endothelium. It also functions in leukocyte-endothelial cell signal transduction, and it may play a role in the development of atherosclerosis and rheumatoid arthritis. VWF is a large multimeric glycoprotein present in blood plasma and produced constitutively in endothelium (in the Weibel-Palade bodies), megakaryocytes (a-granules of platelets), and subendothelial connective tissue. Its primary function is binding to other proteins, particularly Factor VIII and it is important in platelet adhesion to wound sites. WPBs are released from endothelial cells in response to a large number of secretagogues such as thrombin, histamine, peptido-leukotrienes, complement components C5a and C5b-9, superoxide anion, vascular endothelial growth factor (VEGF), sphingosine 1-phosphate, ceramide, purine nucleotides, serotonin, epinephrine, and vasopressin. These agonists of exocytosis can be divided into two distinct groups, those that act by elevating intracellular Ca2+ levels and those that act by raising cAMP levels in the cell. Ca2+-raising agonist like thrombin and histamine are expected to induce a much more vigorous response. It is an important model to explore the mechanism of endothelial cell dysfunction and injury in vitro that thrombin induces endothelial cell exocytosis of WPB.Exocytosis of WPB involves a cycle of budding, docking, priming ,triggering, fusion and recycling. Several sets of proteins mediate exocytosis, including SNAREs, N-ethyl-maleimide-sensitive factor (NSF), Rab, Munc, and other accessory proteins. Soluble NSF attachment protein receptors (SNAREs) are transmembrane proteins that regulate vesicle fusion. Vesicle SNAREs are localized to vesicle membranes, and target SNAREs are localized to target membranes. A set of three SNARE molecules assembles into a stable ternary complex, bringing the vesicle into apposition with a target membrane. SNARE assemble and disassembly funntion importantly in the cycle of vesicle and granule exocytosis. SNARE disassembly is mainly regulated by NSF. NSF regulates vesicle fusion and is inhibited by N-ethyl-maleimide. Each of the three domains of NSF has a specific function. The N-terminal domain of NSF interacts indirectly with SNARE proteins via soluble NSF attachment proteins (SNAP). The D1 domain of NSF hydrolyzes ATP and alters the conformation of the ternary SNARE complex, the D2 domain of NSF that regulates NSF oligomerization. Nitric oxide (NO) is an important molecule that inhibits exocytosis. NO can chemically modify NSF by S-nitrosylating sulfhydryl groups in cysteine residues and prevent disassembly of SNAREs and NSF. Accordingly, elevated production of nitric oxide can inhibit exocytosis. NO is one of the most important molecules synthesized by the endothelial cell. Production of NO results from endothelial NO synthase (eNOS) enzymatic conversion of L-arginine to L-citrulline in the presence of essential cofactors such as FAD, NAD(P)H and tetrahydrobiopterin (BH4). Activation of eNOS is regualated by phosphatidylinositol 3-kinase (PI3 kinase)- serine/threonine protein kinase(Akt) signaling pathway. Akt can phosphorylate eNOS resulting in activation of eNOS. In addition, endothelial dysfunction and injury are closely associated with overexpression of adhesion molecules in endothelial cells. Expression of adhesion molecules is regulated by nuclear factor -κB (NF-κB) signaling pathway. These two genes of VCAM-1 and E-selectin have putative binding sites for NF-κB at their promoter regions to activate gene expression. Under normal condition, NF-κB is sequestrated in cytoplasm bound to inhibitory factor of NF-κB(IκB). When endothelial cells are stimulated by inflammatory mediators such as thrombin, IκB is degraded through complicated upstream molecules. Then NF-κB is released from the NF-κB/IκB complex and translocated from cytoplasm to nucleus, followed by increasing gene expression of cell adhesion molecules such as VCAM-1 and E-selectin. Genipin is a metabolite of geniposide, the major active ingredient of Gardemia jasminoids Ellis fruits, which have long been used in traditional Chinese medicine. This compound has been reported to have anti-inflammatory, anti-diabetic, anti-thrombotic, anti-oxidative, anti-angiogenic and neurotrophic activities. In a variety of animal models, genipin was shown to have anti-inflammatory activities. It significantly inhibited acute inflammation in carrageenan-induced rat paw edema, carrageenan-induced rat air pouch edema and croton oil-induced mouse ear edema models. It also inhibited mouse vascular permeability induced by acetic acid. Genipin may inhibit inflammation in part by inhibiting the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide upon stimulation by lipopolysaccharide/interferon in a murine macrophage cell line, as well as by inhibition of NF-κB activation. However, the molecular mechanisms of anti-inflammatory action of genipin are still not fully understood. Since endothelial exocytosis plays important roles in vascular inflammation, we hypothesized that genipin would exert its anti-inflammation by inhibiting WPB exocytosis from endothelial cells. In this study, we investigated the effects of ginipin on VWF release and P-selecti,E-selectin and VCAM-1 translocation in primary culture of human umbilical vein endothelial cells (HUVECs). We also performed experiments to further examine the mechanisms responsible for the inhibitory effect of endothelial exocytosis induced by this compound.Methods and results1. Treatment with 0.5-16μg/mL genipin had no effect on HUVEC viability Primary human umbilical vein endothelial cell (HUVEC) was isolated from human umbilical cord and cultured The cell viability assessment was performed using Cell Counting Kit-8. HUVECs were treated with 0.5-16μg/mL genipin for 12,24 and 48 h. The results showed that genipin treatment had no effect on HUVEC viability.2. Genipin inhibits thrombin-induced exocytosis of P-selectin, E-selectin, VCAM-1 and VWF from HUVECsHUVECs were pretreated with increasing concentrations of genipin for various times, and then stimulated with 1 U/mL thrombin for 30 min. VWF concentration released in the cell supernatants was measured using VWF ELISA Kit. The amount of P-selectin, E-selectin and VCAM-1 translocation on the cell surface was using cell surface ELISA. Results showed that genipin blocked the exocytosis of P-selectin, E-selectin, VCAM-1 and VWF in dose-dependent manner, with a maximal effect achieved at 8μg/mL. The significant inhibition of genipin was seen within 1 hour of pretreatment and increased in time-dependent way.3.Genipin inhibits mRNA and protein expression of VCAM-1 and E-selectin in the HUVECs induced by thrombinHUVECs were pretreated with increasing concentrations of genipin for various lengths of time and then stimulated with thrombin for 30 min. The mRNA and protein expression of VCAM-1 and E-selectin in the HUVECs was evaluated by real-time RT-PCR and western blot. The results showed that thrombin significantly increased mRNA and protein expression of VCAM-1 and E-selectin in the HUVECs. However, pretreatment with 4-16μg/ml genipin inhibited mRNA and protein expression of VCAM-1 and E-selectin induced by thrombin in dose-dependent manner.4. Genipin inhibits THP-1 cells adhesion to HUVECs induced by thrombinHUVECs were pretreated with 8μg/mL genipin for 1 h and then stimulated with thrombin for 30 min. Fluorescence-labeled THP-1 cells were added to thrombin-stimulated HUVECs and co-cultured for 1 h. After removal of the non-adhesion cells, the remaining adhesion cells were photographed with fluorescence microscopy. The cells were lysed and measured using a spectrofluorometer. Results showed that, after stimulation with thrombine for30 min, adhesion of monocytes to HUVECs significantly increased (P<0.01 compared to normal treatment). When we pretreated HUVECs with 4 and 8μg/mL genipin for 1 h, this compound was found to markedly decrease monocyte adhesion to HUVECs induced by thrombin. (P<0.01 compared to thrombin treatment alone).5. Genipin prolongs bleeding time in miceSince platelet adherence to the vessel wall is mediated by the VWF, P-selectin, VCAM-1 and E-selectin,we predicted that genipin-induced inhibition of endothelial exocytosis would decrease platelet adherence and prolong the bleeding time. Thus, we measured the effect of genipin on the bleeding time in mice to obtain functional evidence that genipin inhibits HUVEC exocytosis in vivo. Anesthetized mice were injected intravenously with various concentrations of genipin or PBS. At various times after drug injection, the distal tip of tail was amputated, and bleeding time was measured. Treatment with PBS had no effect on the bleeding time, with a bleeding time of approximately 6 min. In contrast, genipin dramatically prolonged the bleeding time in dose- dependent manner. At the doses of 5,12.5,25 and 50mg/kg, the bleeding times were 8,10,16 and 18min, respectively. When the dose of the drug increased to 100mg/kg, the bleeding time was more than 20 min. The genipin-induced maximum bleeding time occurred at the 1 hour after the drug treatment, and then the prolonged bleeding time returned to normal level after 1.5 hours of genipin treatment.6. NO mediates genipin-induced inhibition of HUVEC exocytosisNO is an important molecule that inhibits exocytosis, we next examine the ability of genipin to stimulate NO production. We treated HUVECs with various genipin concentrations and with different duration of genipin pretreatment, and then measured the NO level in the cell supernatants. Our results showed that genipin activated HUVEC synthesis of NO in a dose- and time-dependent way.In order to determine whether or not genipin-induced NO production is involved in the exocytosis inhibition by this compound, we examined the effect of L-NAME, an inhibitor of NOS, on genipin-induced exocytosis inhibition. HUVECs were pretreated with various concentrations of L-NAME for 1 h, and then genipin was added for 2 h. The cells were stimulated with thrombin (1U/mL) and VWF level in the supernatants was measured. Our results showed that L-NAME reversed the inhibitory effects of genipin on endothelial exocytosis, suggesting that genipin-induced NO production is involved in its inhibition of endothelial exocytosis.7.Genipin activates PI3K-Akt- eNOS signaling pathway in HUVECsPrevious study showed that genipin inhibited thrombin-induced exocytosis in HUVECs through production of NO. Production of NO resulting from activation of eNOS is regualated by PI3K-Akt- eNOS signaling pathway. Phosphorylation of eNOS by Akt resulted in NO production. Therefore, we investigated the effect of genipin on eNOS activation in order to explore the mechanisms responsible for the inhibitory effect of genipin-induced endothelial exocytosis. HUVECs were treated with different concentrations of genipin or control for various times, and cell lysates were immunoblotted for phospho-eNOS (Ser-1177) or eNOS. Genipin increased phosphorylation level of eNOS in dose and time-dependent manner, but had no effect on the total amount of eNOS within 2 h after drug treatment. These results demonstrated that genipin stimulated eNOS phosphorylation and activated eNOS. We used a pharmacological approach to explore the role of PI3K in genipin activation of eNOS. The LY294002, an inhibitor of PI3K, diminished the ability of genipin to activate phosphorylation of Akt and eNOS. Taken together, these data suggest that genipin activates a pathway that includes PI3K, Akt, and eNOS, and that this pathway mediates genipin inhibition of exocytosis.8. Genipin promotes S-nitrosylation of NSF in HUVECsNO S-nitrosylates sulfhydryl groups of NSF in cysteine residues and prevents disassembly of SNAREs and NSF, leading to decrease of exocytosis. In order to explore effect of genipin on S-nitrosylation of NSF, we used biotin-switch technique (BST), immunoprecipitation and Western blot to determine S-nitrosylation of NSF in HUVECs. Results showed that treatment with 4μg/mL genipin for 1h markedly increased the S-nitrosylation of NSF in HUVECs compared with control group. However, pretreatment with L-NAME(an inhibitor of NOS) or LY294002(an inhibitor of PI3K) inhibited the S-nitrosylation of NSF induced by genipin. All data suggest that genipin activates a pathway that includes PI3K, Akt, and eNOS, increases NO production and promotes S-nitrosylation of NSF in HUVECs. 9.Genipin inhibited thrombin-induced NF-κB signaling pathway activation in HUVECsIn addition, endothelial dysfunction and injury is closely assocated with overexpression of adhesion molecules in endothelial cells. Previous study showed that genipin inhibited the gene expression of VCAM-1 and E-selectin. Expression of adhesion molecules is regulated by nuclear factor -κB (NF-κB) signaling pathway. These two genes of VCAM-1 and E-selectin have putative binding sites for NF-κB at their promoter regions to activate gene expression. Therefore, in this study we explored the effect of genipin on thrombin-induced NF-κB signaling pathway activation using western blotting and immunocytochemistry. Results showed that thrombin significantly increased NF-κB p65 expression in the nuclear extracts compared to normal treatment. In contrast, pretreatment with genipin 8-16μg/mL for 1 h markedly inhibited thrombin-induced increase of nuclear NF-κB p65 in dose-dependent manner. Furthermore, thrombin significantly decreased the IκB expression levels in cytoplasm. However, pretreatment with 8-16μg/mL genipin for1 h markedly inhibited thrombin-induced decrease of IκB expression levels in dose-dependent manner.To confirm the consistency to the result of western blotting, we observed the translocation of NF-κB p65 by using immuocytochemistry.Results showed that thrombin caused NF-κB p65 translocation into nucleus compared to normal treatment. However, pretreatment with 4μg/mL genipin inhibited the translocation of NF-κB p65 from cytoplasm to nucleus. These results were consistent with the result of western blotting.Take together, the conclusions of present study are as follow1. Genipin inhibited exocytosis of P-selectin, E-selectin, VCAM-1 and VWF from HUVECs induce by thrombin.2. Genipin decreased THP-1 cells adhesion to HUVECs induced by thrombin and prolonged the blood time of mice tail vein.3. Genipin activated of PI3K-Akt-eNOS signaling pathway, increases NO production, and induces S-nitrosylation of NSF in HUVECs. 4. Genipin inhibited thrombin-induced HUVEC NF-κB signaling pathway activation, which in turn inhibited the gene and protein expression of adhesion molecules such as VCAM-1 and E-selectin.In conclusion, excessive endothelial exocytosis may contribute to the pathophysiology of inflammation and thrombosis. Genipin, a novel inhibitor of endothelial exocytosis found in this study, may target acute inflammatory events and suppress vascular and endothelial cell inflammatory activation. This compound may represent a new treatment for inflammation and/or thrombosis in which excess exocytosis plays a pathophysiological role.