Objective-Tissue factor (TF) initiates coagulation and indirectly triggers thrombin-dependent protease activated receptor (PAR) signaling. The TF-VIIa complex also directly cleaves PAR2 and promotes angiogenesis in vitro in TF cytoplasmic domain-deleted (TF ␦CT ) mice. Here we address the effect of PAR1 and PAR2 deficiency on angiogenesis in vivo. Methods and Results-In hypoxia-driven angiogenesis of oxygen induced retinopathy (OIR), wild-type, PAR1Ϫ/Ϫ , PAR2 Ϫ/Ϫ , and TF ␦CT mice showed a comparable regression of the superficial vascular plexus during the initial exposure of mice to hyperoxia. However, TF ␦CT mice revascularized areas of central vaso-obliteration significantly faster than wild-type animals. Pharmacological inhibition of the TF-VIIa complex, but not of Xa, and blockade of tyrosine kinase receptor pathways with Gleevec reversed accelerated angiogenesis of TF ␦CT mice to revascularization rates observed in wild-type mice. Genetic deletion of PAR2, but not of PAR1, abolished enhanced revascularization of TF ␦CT mice. PAR1 knock-out animals were indistinguishable from wild-type mice in the model of retinal neoangiogenesis and angiogenesis-dependent subcutaneous tumor growth was unaltered in PAR1-and PAR2-deficient animals. Key Words: tissue factor Ⅲ protease activated receptors Ⅲ angiogenesis Ⅲ coagulation Ⅲ thrombin T issue factor (TF), a transmembrane glycoprotein expressed by vascular and myeloid cells, is the primary cellular initiator of blood coagulation. TF exerts its biological activities by forming a catalytic enzyme complex with coagulation factor VIIa. The TF-VIIa complex then triggers coagulation by binding and activating factor X, leading to thrombin-dependent fibrin deposition and platelet activation. Thrombin can exhibit pleiotrophic effects which are typically mediated through G protein-coupled protease activated receptor (PAR) 1 signaling. 1 The TF-VIIa complex also signals directly by cleaving PAR2, but not PAR1. 2,3 However, the relative contributions of direct TF signaling, downstream coagulation activation, and indirect protease signaling to the underlying pathogenesis of cancer progression and angiogenesis, 3 are incompletely defined in vivo. Conclusion-LossTF deficiency results in vascular failure during development 4 and the proangiogenic phenotype of TF cytoplasmic domain deleted (TF ␦CT ) mice provided direct evidence that TF is involved in angiogenesis. 5 Whether direct TF-VIIa signaling is the only relevant pathway to drive angiogenesis in vivo has not been established. Indeed, thrombin-dependent PAR1 signaling stimulates angiogenesis in certain angiogenesis models in vivo, 6 -8 and the partial embryonic lethality of PAR1 knock-out mice attributable to vascular failure resembles the lethal phenotype of TF deficient animals. 9,10 However, the role of PAR1 in postnatal angiogenesis has not been directly addressed in PAR1 knock-out animals.Thrombin and/or TF-signaling pathways are also involved in tumor angiogenesis, 5,11 but production of proangiogenic factors by tumor cell...

Hyperactivation of NMDA-type glutamate receptors (NMDARs) results in excitotoxicity, contributing to damage in stroke and neurodegenerative disorders. NMDARs are generally comprised of NR1/NR2 subunits but may contain modulatory NR3 subunits. Inclusion of NR3 subunits reduces the amplitude and dramatically decreases the Ca 2ϩ permeability of NMDAR-associated channels in heterologous expression systems and in transgenic mice. Since excessive Ca 2ϩ influx into neurons is a crucial step for excitotoxicity, we asked whether NR3A subunits are neuroprotective. To address this question, we subjected neurons genetically lacking NR3A to various forms of excitotoxic insult. We found that cultured neurons prepared from NR3A knock-out (KO) mice displayed greater sensitivity to damage by NMDA application than wild-type (WT) neurons. In vivo, neonatal, but not adult, WT mice contain NR3A in the cortex, and neonatal NR3A KO mice manifested more damage than WT after hypoxia-ischemia. In adult retina, one location where high levels of NR3A normally persist into adulthood, injection of NMDA into the eye killed more retinal ganglion cells in adult NR3A KO than WT mice. These data suggest that endogenous NR3A is neuroprotective. We next asked whether we could decrease excitotoxicity by overexpressing NR3A. We found that cultured neurons expressing transgenic (TG) NR3A displayed greater resistance to NMDA-mediated neurotoxicity than WT neurons. Similarly in vivo, adult NR3A TG mice subjected to focal cerebral ischemia manifested less damage than WT mice. These data suggest that endogenous NR3A protects neurons, and exogenously added NR3A increases neuroprotection and could be potentially exploited as a therapeutic.

Gene expression profiles in laser-induced glaucomatous monkey retinas were determined, and only a very small population of genes was up- or downregulated in glaucomatous eyes. Upregulation of ceruloplasmin protein was found in the Müller cells.

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IntroductionDisseminated intravascular coagulation driven by tissue factor (TF) is associated with systemic inflammatory disorders, including viral hemorrhagic fevers and severe sepsis (for reviews, see Riewald and Ruf 1 and Ruf 2 ). Inhibition of the TF pathway by potent TF-directed inhibitors reduced inflammatory cytokine levels and increased survival in lethal models of bacterial sepsis in primates 3-5 and attenuated acute lung injury. 6 Although coagulation blockade is likely to be beneficial in sepsis by reducing microthrombosis, subsequent hypoxic organ dysfunction, and inflammation, blocking thrombin generation per se has poor efficacy in prolonging survival in primate models. 7,8 Therefore, it remains of interest to understand how signaling of the TF pathway makes contributions to sepsis mortality. Genetic reduction of TF or VIIa levels in mice increased survival in endotoxemia, but protease activated receptor 1 (PAR1) or PAR2 deficiency alone or in combination did not reduce lethality. [9][10][11] Whether the TF pathway contributes to severe systemic inflammation through signaling thus remains incompletely understood.In the response to microbial pathogen challenge or lipopolysaccharide (LPS)-induced toll-like receptor 4 (TLR4) activation, cells of the monocyte/macrophage lineage are the primary cell types that up-regulate TF to trigger intravascular coagulation. [12][13][14][15] However, the subsequent crosstalk between coagulation activation and inflammation is complex. Thrombin and PAR signaling may amplify the coagulant response by further inducing TF in endothelial cells [16][17][18] or attenuate coagulation and inflammation through the activated protein C (PC)/endothelial cell PC receptor (EPCR) pathway that uses PARs as signaling receptors. [19][20][21][22] In addition, endotoxin challenge of TF cytoplasmic domain-deleted (TF ⌬CT ) mice indicated that TF can regulate leukocyte rolling and recruitment, presumably by altering vessel wall properties. 23 The TF cytoplasmic domain also regulates integrin activation and cell migration 24,25 and has roles in PAR-2-dependent angiogenesis. 26 Here, we identify a novel role of the TF cytoplasmic domain in regulating the procoagulant response induced by TLR signaling in macrophages. LPS activation of macrophages is mediated by a receptor complex of CD14 and TLR4. 27 The signaling pathways by which LPS induces TF mRNA expression in monocytes have been studied in detail, 28 but maturation of monocytes into macrophages promotes diverse phenotypic changes, including the expression of TF 29 as well as the TF-VIIa signaling receptor PAR2. 30 We find in macrophages that LPS-mediated TF up-regulation is controlled by the TF cytoplasmic domain through p38-dependent regulation of the ERK1/2 MAP kinase pathway. However, regulation of TF expression is independent of PAR2 signaling and specific for TLR, but not interferon ␥, signaling. The data provide new evidence for an autoregulatory role of the TF cytoplasmic domain to control procoagulant responses in endotoxemia. M...