Nicole C. Kaneider

'Role reversal' for the receptor PAR1 in sepsis-induced vascular damage

Sepsis is a deadly disease characterized by considerable derangement of the proinflammatory, anti-inflammatory and coagulation responses. Protease-activated receptor 1 (PAR1), an important regulator of endothelial barrier function and blood coagulation, has been proposed to be involved in the lethal sequelae of sepsis, but it is unknown whether activation of PAR1 is beneficial or harmful. Using a cell-penetrating peptide (pepducin) approach, we provide evidence that PAR1 switched from being a vascular-disruptive receptor to a vascular-protective receptor during the progression of sepsis in mice. Unexpectedly, we found that the protective effects of PAR1 required transactivation of PAR2 signaling pathways. Our results suggest therapeutics that selectively activate PAR1-PAR2 complexes may be beneficial in the treatment of sepsis.

Blocking the Protease-Activated Receptor 1-4 Heterodimer in Platelet-Mediated Thrombosis

Background— Thrombin is the most potent agonist of platelets and plays a critical role in the development of arterial thrombosis. Human platelets express dual thrombin receptors, protease-activated receptor (PAR) 1 and PAR4; however, there are no therapeutic strategies that effectively target both receptors. Methods and Results— Platelet aggregation studies demonstrated that PAR4 activity is markedly enhanced by thrombin–PAR1 interactions. A combination of bivalirudin (hirulog) plus a novel PAR4 pepducin antagonist, P4pal-i1, effectively inhibited aggregation of human platelets to even high concentrations of thrombin and prevented occlusion of carotid arteries in guinea pigs. Likewise, combined inhibition of PAR1 and PAR4 with small-molecule antagonists and pepducins was effective against carotid artery occlusion. Coimmunoprecipitation and fluorescence resonance energy transfer studies revealed that PAR1 and PAR4 associate as a heterodimeric complex in human platelets and fibroblasts. PAR1-PAR4 cofactoring was shown by acceleration of thrombin cleavage and signaling of PAR4 on coexpression with PAR1. Conclusions— We show that PAR1 and PAR4 form a stable heterodimer that enables thrombin to act as a bivalent functional agonist. These studies suggest that targeting the PAR1-PAR4 complex may present a novel therapeutic opportunity to prevent arterial thrombosis.

Platelet Matrix Metalloprotease-1 Mediates Thrombogenesis by Activating PAR1 at a Cryptic Ligand Site

Matrix metalloproteases (MMPs) play important roles in normal and pathological remodeling processes including atherothrombotic disease, inflammation, angiogenesis, and cancer. MMPs have been viewed as matrix-degrading enzymes, but recent studies have shown that they possess direct signaling capabilities. Platelets harbor several MMPs that modulate hemostatic function and platelet survival; however their mode of action remains unknown. We show that platelet MMP-1 activates protease-activated receptor-1 (PAR1) on the surface of platelets. Exposure of platelets to fibrillar collagen converts the surface-bound proMMP-1 zymogen to active MMP-1, which promotes aggregation through PAR1. Unexpectedly, MMP-1 cleaves PAR1 at a distinct site that strongly activates Rho-GTP pathways, cell shape change and motility, and MAPK signaling. Blockade of MMP1-PAR1 curtails thrombogenesis under arterial flow conditions and inhibits thrombosis in animals. These studies provide a link between matrix-dependent activation of metalloproteases and platelet-G protein signaling and identify MMP1-PAR1 as a potential target for the prevention of arterial thrombosis.

Reversing systemic inflammatory response syndrome with chemokine receptor pepducins

We describe a new therapeutic approach for the treatment of lethal sepsis using cell-penetrating lipopeptides—termed pepducins—that target either individual or multiple chemokine receptors. Interleukin-8 (IL-8), a ligand for the CXCR1 and CXCR2 receptors, is the most potent endogenous proinflammatory chemokine in sepsis. IL-8 levels rise in blood and lung fluids to activate neutrophils and other cells, and correlate with shock, lung injury and high mortality. We show that pepducins derived from either the i1 or i3 intracellular loops of CXCR1 and CXCR2 prevent the IL-8 response of both receptors and reverse the lethal sequelae of sepsis, including disseminated intravascular coagulation and multi-organ failure in mice. Conversely, pepducins selective for CXCR4 cause a massive leukocytosis that does not affect survival. CXCR1 and CXCR2 pepducins conferred nearly 100% survival even when treatment was postponed, suggesting that our approach might be beneficial in the setting of advanced disease.

The immunomodulator FTY720 interferes with effector functions of human monocyte-derived dendritic cells

The potent immunomodulator FTY720 elicits immunosuppression via acting on sphingosine 1‐phosphate receptors (S1PR), thereby leading to an entrapment of lymphocytes in the secondary lymphoid tissue. To elucidate the potential in vitro effects of this drug on human monocyte‐derived DC, we used low nanomolar therapeutic concentrations of FTY720 and phosphorylated FTY720 (FTY720‐P) and investigated their influence on DC surface marker expression, protein levels of S1PR and DC effector functions: antigen uptake, chemotaxis, cytokine production, allostimulatory and Th‐priming capacity. We report that both FTY720 and FTY720‐P reduce chemotaxis of immature and mature DC. Mature DC generated in the presence of FTY720 or FTY720‐P showed an impaired immunostimmulatory capacity and reduced IL‐12 but increased IL‐10 production. T cells cultured in the presence of FTY720‐ or FTY720‐P‐treated DC showed an altered cytokine production profile indicating a shift from Th1 toward Th2 differentiation. In treated immature and mature DC, expression levels for two S1PR proteins, S1P1 and S1P4, were reduced. We conclude that in vitro treatment with FTY720 affects DC features that are essential for serving their role as antigen‐presenting cells. This might represent a new aspect of the overall immunosuppressive action of FTY720 and makes DC potential targets of further sphingolipid‐derived drugs.

Targeting a metalloprotease-PAR1 signaling system with cell-penetrating pepducins inhibits angiogenesis, ascites, and progression of ovarian cancer

Gene chip and proteomic analyses of tumors and stromal tissue has led to the identification of dozens of candidate tumor and host components potentially involved in tumor-stromal interactions, angiogenesis, and progression of invasive disease. In particular, matrix metalloproteases (MMP) have emerged as important biomarkers and prognostic factors for invasive and metastatic cancers. From an initial screen of benign versus malignant patient fluids, we delineated a metalloprotease cascade comprising MMP-14, MMP-9, and MMP-1 that culminates in activation of PAR1, a G protein-coupled protease-activated receptor up-regulated in diverse cancers. In xenograft models of advanced peritoneal ovarian cancer, PAR1-dependent angiogenesis, ascites formation, and metastasis were effectively inhibited by i.p. administration of cell-penetrating pepducins based on the intracellular loops of PAR1. These data provide an in vivo proof-of-concept that targeting the metalloprotease-PAR1 signaling system may be a novel therapeutic approach in the treatment of ovarian cancer. [Mol Cancer Ther 2008;7(9):2746–57]

Migration of human monocytes in response to procalcitonin.

ObjectiveCirculating serum levels of procalcitonin rise significantly during bacterial infection. Because calcitonin is known to be a monocyte chemoattractant, we investigated whether procalcitonin, a prohormone of calcitonin, also affects leukocyte migration. DesignProspective, controlled in vitro study. SettingUniversity research laboratories. InterventionsForearm venous blood polymorphonuclear neutrophils and monocytes were isolated from healthy human donors. Cell migration was assessed in a blindwell chemotaxis chamber. The distance of migration into filter micropores was measured. To biochemically confirm functional data on cell migration, effects of procalcitonin on cellular levels of cyclic adenosine monophosphate were measured by high-performance liquid chromatography. Measurements and Main ResultsBoth procalcitonin and calcitonin elicited dose-dependent migration of monocytes at concentrations from the femtomolar to the micromolar range. Neutrophils did not migrate toward procalcitonin or calcitonin, nor was their oxygen free radical release affected as measured fluorimetrically. Checkerboard analysis of monocyte locomotion revealed procalcitonin-induced migration as true chemotaxis. Pretreatment of monocytes with procalcitonin or calcitonin rapidly deactivated their migratory response to formyl-Met-Leu-Phe, and both also induced homologous deactivation of migration. Procalcitonin elevated levels of cyclic adenosine monophosphate in monocytes. ConclusionsIn vitro procalcitonin is a monocyte chemoattractant that deactivates chemotaxis in the presence of additional inflammatory mediators. Procalcitonin stimulates cyclic adenosine monophosphate production in monocytes, suggesting that its action may be specific and comparable with calcitonin, which exerts similar functions.

The Neuropeptide Secretoneurin Acts as a Direct Angiogenic Cytokine In Vitro and In Vivo

Background—Secretoneurin is an abundant neuropeptide of the central, peripheral, and autonomic nervous systems, located in nerve fibers characterized by a close interaction with blood vessels and known to stimulate endothelial cell migration. Methods and Results—We hypothesized that secretoneurin might act as an angiogenic cytokine and tested for these effects in vivo using a mouse cornea neovascularization model and in vitro by assessing capillary tube formation in a matrigel assay. In vivo, secretoneurin-induced neovasculature is characterized by a distinct pattern of arterial and venous vessels of large diameter and length. Immunohistochemical staining for CD-31 revealed endothelial lining of the inner surface of these vessels, and recruitment of &agr;-smooth muscle actin–positive perivascular cells suggests vessel maturation. In vitro, secretoneurin-induced capillary tube formation was dose dependent and specific, confirming that effects of secretoneurin occur directly on endothelial cells. Secretoneurin also stimulated proliferation and exerted antiapoptotic effects on endothelial cells and activated intracellular phosphatidylinositol 3′ kinase/Akt and mitogen-activated protein kinase pathways, as demonstrated by increased phosphorylation of Akt and extracellular signal–regulated kinase. Conclusions—These data show that secretoneurin represents a novel direct angiogenic cytokine and reiterate the coordinated relationship between nervous and vascular systems.

A matrix metalloprotease-PAR1 system regulates vascular integrity, systemic inflammation and death in sepsis.

Sepsis is a deadly disease characterized by the inability to regulate the inflammatory–coagulation response in which the endothelium plays a key role. The cause of this perturbation remains poorly understood and has hampered the development of effective therapeutics. Matrix metalloproteases (MMPs) are involved in the host response to pathogens, but can also cause uncontrolled tissue damage and contribute to mortality. We found that human sepsis patients had markedly elevated plasma proMMP‐1 and active MMP‐1 levels, which correlated with death at 7 and 28 days after diagnosis. Likewise, septic mice had increased plasma levels of the MMP‐1 ortholog, MMP‐1a. We identified mouse MMP‐1a as an agonist of protease‐activated receptor‐1 (PAR1) on endothelial cells. MMP‐1a was released from endothelial cells in septic mice. Blockade of MMP‐1 activity suppressed endothelial barrier disruption, disseminated intravascular coagulation (DIC), lung vascular permeability as well as the cytokine storm and improved survival, which was lost in PAR1‐deficient mice. Infusion of human MMP‐1 increased lung vascular permeability in normal wild‐type mice but not in PAR1‐deficient mice. These findings implicate MMP‐1 as an important activator of PAR1 in sepsis and suggest that therapeutics that target MMP1‐PAR1 may prove beneficial in the treatment of sepsis.

ER stress transcription factor Xbp1 suppresses intestinal tumorigenesis and directs intestinal stem cells

X-box–binding protein 1 suppresses tumor formation in the gut by regulating Ire1α and Stat3-mediated regenerative responses in the epithelium as a consequence of ER stress.