Jian Guo Geng

Andrographolide attenuates inflammation by inhibition of NF-kappa B activation through covalent modification of reduced cysteine 62 of p50.

NF-κB is a central transcriptional factor and a pleiotropic regulator of many genes involved in immunological responses. During the screening of a plant extract library of traditional Chinese herbal medicines, we found that NF-κB activity was potently inhibited by andrographolide (Andro), an abundant component of the plant Andrographis that has been commonly used as a folk remedy for alleviation of inflammatory disorders in Asia for millennia. Mechanistically, it formed a covalent adduct with reduced cysteine (62) of p50, thus blocking the binding of NF-κB oligonucleotide to nuclear proteins. Andro suppressed the activation of NF-κB in stimulated endothelial cells, which reduced the expression of cell adhesion molecule E-selectin and prevented E-selectin-mediated leukocyte adhesion under flow. It also abrogated the cytokine- and endotoxin-induced peritoneal deposition of neutrophils, attenuated septic shock, and prevented allergic lung inflammation in vivo. Notably, it had no suppressive effect on IκBα degradation, p50 and p65 nuclear translocation, or cell growth rates. Our results thus reveal a unique pharmacological mechanism of Andro’s protective anti-inflammatory actions.

HDAC5 is a repressor of angiogenesis and determines the angiogenic gene expression pattern of endothelial cells

Class IIa histone deacetylases (HDACs) are signal-responsive regulators of gene expression involved in vascular homeostasis. To investigate the differential role of class IIa HDACs for the regulation of angiogenesis, we used siRNA to specifically suppress the individual HDAC isoenzymes. Silencing of HDAC5 exhibited a unique pro-angiogenic effect evidenced by increased endothelial cell migration, sprouting, and tube formation. Consistently, overexpression of HDAC5 decreased sprout formation, indicating that HDAC5 is a negative regulator of angiogenesis. The antiangiogenic activity of HDAC5 was independent of myocyte enhancer factor-2 binding and its deacetylase activity but required a nuclear localization indicating that HDAC5 might affect the transcriptional regulation of gene expression. To identify putative HDAC5 targets, we performed microarray expression analysis. Silencing of HDAC5 increased the expression of fibroblast growth factor 2 (FGF2) and angiogenic guidance factors, including Slit2. Antagonization of FGF2 or Slit2 reduced sprout induction in response to HDAC5 siRNA. Chromatin immunoprecipitation assays demonstrate that HDAC5 binds to the promoter of FGF2 and Slit2. In summary, HDAC5 represses angiogenic genes, such as FGF2 and Slit2, which causally contribute to capillary-like sprouting of endothelial cells. The derepression of angiogenic genes by HDAC5 inactivation may provide a useful therapeutic target for induction of angiogenesis.

Repulsive axon guidance molecule Slit3 is a novel angiogenic factor.

Slits are large, secreted repulsive axon guidance molecules. Recent genetic studies revealed that the Slit3 is dispensable for neural development but required for non-neuron-related developmental processes, such as the genesis of the diaphragm and kidney. Here we report that Slit3 potently promotes angiogenesis, a process essential for proper organogenesis during embryonic development. We observed that Slit3 is expressed and secreted by both endothelial cells and vascular smooth muscle cells in vasculature and that the Slit cognate receptors Robo1 and Robo4 are universally expressed by endothelial cells, suggesting that Slit3 may act in paracrine and autocrine manners to regulate endothelial cells. Cellular function studies revealed that Slit3 stimulates endothelial-cell proliferation, promotes endothelial-cell motility and chemotaxis via interaction with Robo4, and accelerates endothelial-cell vascular network formation in vitro with a specific activity comparable with vascular endothelial growth factor. Furthermore, Slit3 stimulates neovessel sprouting ex vivo and new blood vessel growth in vivo. Consistent with these observations, the Slit3 knockout mice display disrupted angiogenesis during embryogenesis. Taken together, our studies reveal that the repulsive axon guidance molecule Slit3 is a novel and potent angiogenic factor and functions to promote angiogenesis in coordinating organogenesis during embryonic development.

NF-κB Transcription Factor p50 Critically Regulates Tissue Factor in Deep Vein Thrombosis

NF-κB transcription factors regulate the expression of tissue factor (TF), a principal initiator of the coagulation cascade. Dominant among them is the p50/p65 heterodimer. Here we report that Andrographolide (Andro; a p50 inhibitor) and genetic deletion of p50 attenuated TF activity in stimulated endothelial cells and monocytes/macrophages. Results of the electrophoretic mobility “supershift” assay and chromatin immunoprecipitation demonstrated the direct interaction of the p50/p65 heterodimer with the NF-κB site of the human TF promoter. Andro-treated and p50 null mice both exhibited blunted TF expression and reduced venous thrombosis, which were recapitulated by an anti-murine TF antibody in vivo. Our findings thus indicate that regulation of TF by NF-κB transcription factor p50 is essential for the pathogenesis of deep vein thrombosis and suggest that specific inhibitors of p50, such as Andro, may be therapeutically valuable for preventing and perhaps treating venous thrombosis.

N-desulfated non-anticoagulant heparin inhibits leukocyte adhesion and transmigration in vitro and attenuates acute peritonitis and ischemia and reperfusion injury in vivo

Abstract.Objective: Heparin, a highly sulfated proteoglycan, is known to have strong anticoagulant and anti-inflammatory activities. Here we sought to generate a heparin derivative, which had a significantly lower anticoagulant activity while retaining its strong anti-inflammatory activity.¶Materials and methods: Heparin was chemically modified and this discrete set of the heparin derivatives was tested for their anticoagulant activities, such as activated partial thromboplastin time (APTT), and anti-inflammatory activities, such as leukocyte adhesion and transmigration in vitro and acute peritonitis and ischemia and reperfusion injury in vivo.¶Results: We found that an N-desulfated heparin had 188-fold (compared to heparin) and 32-fold (compared to low molecular weight heparin; LMWH) reductions of APTT. The N-desulfated heparin inhibited adhesion of human promyeloid HL-60 cells to the stimulated human umbilical vein endothelial cells (HUVECs) under a physiological shear stress. It also prevented the transmigration of human neutrophils through the monolayers of the stimulated HUVECs. Further, intravenous administration of this compound attenuated the peritoneal infiltration of neutrophils in a mouse model of acute peritonitis, and reduced tissue edema and leukocyte deposition in a rabbit ear model of ischemia and reperfusion injury.¶Conclusion: It is to our best knowledge that the N-desulfated heparin has the lowest anticoagulant activity among LMWH and chemically modified heparin derivatives, while preserving a potent anti-inflammatory activity. These combined properties appear to suggest it as a safer medicine for treatment of inflammation.

Heparan sulfate-like proteoglycans mediate adhesion of human malignant melanoma A375 cells to P-selectin under flow.

Selectins, a family of cell adhesion molecules, bind to sialylated and fucosylated carbohydrates, such as sialyl Lewisx (SLex) and its derivatives, as their minimal recognition motif. Here we report that P-selectin bound to human malignant melanoma A375 cells and mediated their adhesion under flow. However, probing with a specific Ab failed to detect any apparent expression of SLex. This finding was bolstered by reduced expression of α-1,3-fucosyltransferase VII mRNA and by absence of the cell surface expression of P-selectin glycoprotein ligand-1. Instead, they expressed heparan sulfate-like proteoglycans on their cell surfaces. Treatment with β-d-xyloside (a proteoglycan biosynthesis inhibitor) or heparinases could reduce the binding of these cells to P-selectin. In the competition assays, heparin, but not other proteoglycans, could abolish the P-selectin recognition. Further, we found that P-selectin could bind specifically to human tongue squamous cancer Tca-8113 cells, which had negative staining of SLex but positive staining of heparan sulfates. Both β-d-xyloside and heparinases could reduce the binding of P-selectin to Tca-8113 cells. Our results thus indicate that heparan sulfate-like proteoglycans can mediate adhesion of certain types of non-blood borne, “epithelial-like” human cancer cells to P-selectin.

Slit-Robo signaling mediates lymphangiogenesis and promotes tumor lymphatic metastasis

The Slit family of guidance cues binds to Roundabout (Robo) receptors to modulate neuronal, leukocytic, and endothelial migration. Slit-Robo signaling had been reported to function as chemoattractive signal for vascular endothelial cells during angiogenesis. In this study, we found that Robo1 was expressed in lymphatic endothelial cells to mediate the migration and tube formation of these cells upon Slit2 stimulation, which were specifically inhibited by the function-blocking antibody R5 to Slit2/Robo1 interaction. To further explore the lymphangiogenic effect and significance mediated by Slit-Robo signaling, we intercrossed Slit2 transgenic mice with a non-metastatic RIP1-Tag2 mouse tumor model, and found that transgenic overexpression of Slit2 significantly enhanced tumor lymphangiogenesis and subsequently promoted mesenteric lymph node metastasis of pancreatic islet tumors. Taken together, our findings reveal that through interacting with Robo1, Slit2 is a novel and potent lymphangiogenic factor and contributes to tumor lymphatic metastasis.

Slit2 Regulates Attractive Eosinophil and Repulsive Neutrophil Chemotaxis through Differential srGAP1 Expression during Lung Inflammation

Directional migration of leukocytes is an essential step in leukocyte trafficking during inflammatory responses. However, the molecular mechanisms governing directional chemotaxis of leukocytes remain poorly understood. The Slit family of guidance cues has been implicated for inhibition of leuocyte migration. We report that Clara cells in the bronchial epithelium secreted Slit2, whereas eosinophils and neutrophils expressed its cell-surface receptor, Robo1. Compared to neutrophils, eosinophils exhibited a significantly lower level of Slit-Robo GTPase-activating protein 1 (srGAP1), leading to activation of Cdc42, recruitment of PI3K to Robo1, enhancment of eotaxin-induced eosinophil chemotaxis, and exaggeration of allergic airway inflammation. Notably, OVA sensitization elicited a Slit2 gradient at so-called bronchus–alveoli axis, with a higher level of Slit2 in the bronchial epithelium and a lower level in the alveolar tissue. Aerosol administration of rSlit2 accelerated eosinophil infiltration, whereas i.v. administered Slit2 reduced eosinophil deposition. In contrast, Slit2 inactivated Cdc42 and suppressed stromal cell-derived factor-1α–induced chemotaxis of neutrophils for inhibiting endotoxin-induced lung inflammation, which were reversed by blockade of srGAP1 binding to Robo1. These results indicate that the newly identified Slit2 gradient at the bronchus–alveoli axis induces attractive PI3K signaling in eosinophils and repulsive srGAP1 signaling in neutrophils through differential srGAP1 expression during lung inflammation.

IFN Regulatory Factor-1 Is Required for the Up-Regulation of the CD40-NF-κB Activator 1 Axis During Airway Inflammation

Recent studies show that NF-κB activator 1 (Act1) functions as an important adapter molecule for CD40-mediated signaling in epithelial cells. To explore the physiological function of the CD40-Act1 axis, we studied the regulation of gene expression of CD40 and Act1 both in vivo and in cell culture models. Although CD40 and Act1 are up-regulated in mouse lung upon LPS stimulation, IL-1 plus IFN-α, -β, or -γ synergistically up-regulate both CD40 and Act1 gene expression in human epithelial A549 cells. Cycloheximide superinduces the Act1 mRNA, whereas actinomycin D completely abolishes the Act1 mRNA, indicating that the induction of Act1 mRNA is at the transcriptional level and does not require protein synthesis. Promoter sequence analyses identified putative IFN regulatory factor (IRF)-1, C/EBP-β, and AP-1 transcription factor binding sites in the Act1 promoter. Although mutation of any of the three sites abolished the promoter activity, Abs against IRF-1 and C/EBP-β, but not AP-1, blocked the formation of the DNA-binding complex induced by IL-1 plus IFN-β stimulation, suggesting cooperative action between IRF-1 and C/EBP-β in mediating Act1 promoter activity. Importantly, LPS-induced gene expression of CD40 and Act1 in the mouse lung is abolished in IRF-1−/− mice, indicating an essential role of transcription factor IRF-1 in the coordinated regulation of these two genes during airway inflammation. The induced expression of the CD40-Act1 axis by inflammatory cytokines in epithelial cells probably plays an important role in priming these cells for their response to CD40 ligand during airway inflammation.

P-selectin Cross-links PSGL-1 and Enhances Neutrophil Adhesion to Fibrinogen and ICAM-1 in a Src Kinase-Dependent, but GPCR-Independent Mechanism

Endothelial and platelet P-selectin (CD62P) and leukocyte integrin αMβ2 (CD11bCD18, Mac-1) are cell adhesion molecules essential for host defense and innate immunity. Upon inflammatory challenges, P-selectin binds to PSGL-1 (P-selectin glycoprotein ligand-1, CD162) to mediate neutrophil rolling, during which integrins become activated by extracellular stimuli for their firm adhesion in a G-protein coupled receptor (GPCR)-dependent mechanism. Here we show that cross-linking of PSGL-1 by dimeric or multimeric forms of platelet P-selectin, P-selectin receptor-globulin, anti-PSGL-1 mAb and its F(ab’)2 induced adhesion of human neutrophils to fibrinogen (Fg) and intercellular cell adhesion molecule-1 (ICAM-1, CD54) and triggered a moderate clustering of αMβ2, but monomeric forms of soluble P-selectin and anti-PSGL-1 Fab did not. Interestingly, P-selectin did not induce a detectable interleukine-8 (IL-8) secretion (<0.1 ng/ml) in 30 minutes, whereas a high concentration of IL-8 (>50 ng/ml) was required to increase neutrophil adhesion to Fg. P-selectin-induced neutrophil adhesion was significantly inhibited by PP2 (a Src kinase inhibitor), but not by Pertussis toxin (PTX; a GPCR inhibitor). Activated platelets also increased neutrophil binding to fibrinogen and triggered tyrosine phosphorylation of cellular proteins. Our results indicate that P-selectin-induced integrin activation (Src kinase-dependent) is distinct from that elicited by cytokines, chemokines, chemoattractants (GPCR-dependent), suggesting that these two signal transduction pathways may cooperate for maximal activation of leukocyte integrins.