No abstract

As do cytokine receptors and receptor tyrosine kinases, G protein-coupled receptors (GPCRs) signal to Janus kinases (Jaks) and signal transducers and activators of transcription (STATs). However, the early biochemical events linking GPCRs to this signaling pathway have been unclear. Here we show that GPCRstimulated Rac activity and the subsequent generation of reactive oxygen species are necessary for activating tyrosine phosphorylation of Jaks and STAT-dependent transcription. The requirement for Rac activity can be overcome by addition of hydrogen peroxide. Expression of activated mutants of Rac1 is sufficient to activate Jak2 and STAT-dependent transcription, and the activation of Jak2 correlates with the ability of Rac1 to bind to NADPH oxidase subunit p67 phox . We further show that GPCR agonists stimulate tyrosine phosphorylation of STAT1 and STAT3 proteins in a Rac-dependent manner. The tyrosine phosphorylation of STAT3 is biphasic; the first peak of phosphorylation is weak and correlates with rapid activation of Jaks by GPCRs, whereas the second peak is stronger and requires the synthesis of an autocrine factor. Rho also plays an essential role in the induction of STAT transcriptional activity. Our results highlight a novel role for Rho GTPases in mediating the regulatory effects of GPCRs on STAT-dependent gene expression.

Preterm birth (PTB) is firmly linked to inflammation regardless of the presence of infection. Proinflammatory cytokines, including IL-1β, are produced in gestational tissues and can locally upregulate uterine activation proteins. Premature activation of the uterus by inflammation may lead to PTB, and IL-1 has been identified as a key inducer of this condition. However, all currently available IL-1 inhibitors are large molecules that exhibit competitive antagonism properties by inhibiting all IL-1R signaling, including transcription factor NF-κB, which conveys important physiological roles. We hereby demonstrate the efficacy of a small noncompetitive (all-d peptide) IL-1R–biased ligand, termed rytvela (labeled 101.10) in delaying IL-1β–, TLR2-, and TLR4-induced PTB in mice. The 101.10 acts without significant inhibition of NF-κB, and instead selectively inhibits IL-1R downstream stress-associated protein kinases/transcription factor c-jun and Rho GTPase/Rho-associated coiled-coil–containing protein kinase signaling pathways. The 101.10 is effective at decreasing proinflammatory and/or prolabor genes in myometrium tissue and circulating leukocytes in all PTB models independently of NF-κB, undermining NF-κB role in preterm labor. In this work, biased signaling modulation of IL-1R by 101.10 uncovers a novel strategy to prevent PTB without inhibiting NF-κB.

Angiogenesis of the microvasculature is central to the etiology of many diseases including proliferative retinopathy, age-related macular degeneration and cancer. A mouse model of microvascular angiogenesis would be very valuable and enable access to a wide range of genetically manipulated tissues that closely approximate small blood vessel growth in vivo. Vascular endothelial cells cultured in vitro are widely used, however, isolating pure vascular murine endothelial cells is technically challenging. A microvascular mouse explant model that is robust, quantitative and can be reproduced without difficulty would overcome these limitations. Here we characterized and optimized for reproducibility an organotypic microvascular angiogenesis mouse and rat model from the choroid, a microvascular bed in the posterior of eye. The choroidal tissues from C57BL/6J and 129S6/SvEvTac mice and Sprague Dawley rats were isolated and incubated in Matrigel. Vascular sprouting was comparable between choroid samples obtained from different animals of the same genetic background. The sprouting area, normalized to controls, was highly reproducible between independent experiments. We developed a semi-automated macro in ImageJ software to allow for more efficient quantification of sprouting area. Isolated choroid explants responded to manipulation of the external environment while maintaining the local interactions of endothelial cells with neighboring cells, including pericytes and macrophages as evidenced by immunohistochemistry and fluorescence-activated cell sorting (FACS) analysis. This reproducible ex vivo angiogenesis assay can be used to evaluate angiogenic potential of pharmacologic compounds on microvessels and can take advantage of genetically manipulated mouse tissue for microvascular disease research.

Retinopathy of prematurity (ROP), an ocular disease characterized by the onset of vascular abnormalities in the developing retina, is the major cause of visual impairment and blindness in premature neonates. ROP is a complex condition in which various factors participate at different stages of the disease leading to microvascular degeneration followed by neovascularization, which in turn predisposes to retinal detachment. Current ablative therapies (cryotherapy and laser photocoagulation) used in the clinic for the treatment of ROP have limitations and patients can still have long-term effects even after successful treatment. New treatment modalities are still emerging. The most promising are the therapies directed against VEGF; more recently the use of preventive dietary supplementation with ω-3 polyunsaturated fatty acid may also be promising. Other than pharmacologic and nutritional approaches, cell-based strategies for vascular repair are likely to arise from advances in regenerative medicine using stem cells. In addition to all of these, a greater understanding of other factors involved in regulating pathologic retinal angiogenesis continues to emerge, suggesting potential targets for therapeutic approaches. This review summarizes an update on the current state of knowledge on ROP from our and other laboratories, with particular focus on the role of nitro-oxidative stress and notably trans-arachidonic acids in microvascular degeneration, semaphorin 3 operating as vasorepulsive molecules in the avascular hypoxic retina and in turn impairing revascularization, succinate and its receptor GPR91 in neuron-mediated retinal neovascularization, and ω-3 lipids as modulators of preretinal neovascularization.

Objective-Prompt post-hypoxia-ischemia (HI) revascularization has been suggested to improve outcome in adults and newborn subjects. Other than hypoxia-inducible factor, sensors of metabolic demand remain largely unknown. During HI, anaerobic respiration is arrested resulting in accumulation of carbohydrate metabolic intermediates. As such succinate readily increases, exerting its biological effects via a specific receptor, G-protein-coupled receptor (GPR) 91. We postulate that succinate/GPR91 enhances post-HI vascularization and reduces infarct size in a model of newborn HI brain injury. Approach and Results-The Rice-Vannucci model of neonatal HI was used. Succinate was measured by mass spectrometry, and microvascular density was evaluated by quantification of lectin-stained cryosection. Gene expression was evaluated by real-time polymerase chain reaction. Succinate levels rapidly increased in the penumbral region of brain infarcts. GPR91 was foremost localized not only in neurons but also in astrocytes. Microvascular density increased at 96 hours after injury in wild-type animals; it was diminished in GPR91-null mice leading to an increased infarct size. Stimulation with succinate led to an increase in growth factors implicated in angiogenesis only in wild-type mice. To explain the mode of action of succinate/GPR91, we investigated the role of prostaglandin E 2 -prostaglandin E receptor 4, previously proposed in neural angiogenesis. Succinate-induced vascular endothelial growth factor expression was abrogated by a cyclooxygenase inhibitor and a selective prostaglandin E receptor 4 antagonist. This antagonist also abolished succinate-induced neovascularization. Conclusions-We uncover a dominant metabolic sensor responsible for post-HI neurovascular adaptation, notably succinate/ GPR91, acting via prostaglandin E 2 -prostaglandin E receptor 4 to govern expression of major angiogenic factors. We propose that pharmacological intervention targeting GPR91 could improve post-HI brain recovery. [20][21][22] Yet, their mode of action has generally remained inexplicable, until membrane-bound receptors that are specifically activated by these metabolites have been identified. 23 In this context, succinate has been shown to contribute to the developmental and possibly aberrant retinal neovascularization presumably through actions on G-protein-coupled receptor (GPR) 91 independent of hypoxia-inducible factor activation 24 ; accordingly, siRNAand shRNA-GPR91 interfere with developmental angiogenesis and partly with abnormal preretinal neovascularization. Nonstandard Abbreviations and Acronyms EP4prostaglandin E receptor 4 GPR G-protein-coupled receptor HI hypoxia-ischemia VEGF Vascular endothelial growth factor Figure 1. Succinate accumulates in the penumbral region after cerebral hypoxia-ischemia (HI) where G-protein-coupled receptor (GPR) 91 is expressed in neurons and astrocytes. A, GPR91 colocalizes with NeuN (i) and glial fibrillary acidic protein (GFAP) (ii) in the rat cerebral cortex. Rabbit anti-GPR91 was used to l...

Purpose – The purpose of this article is to propose a theoretical model explaining information technology outsourcing performance in the public sector as well as a set of empirically testable propositions to improve the understanding of key determinants of success. Design/methodology/approach – Based on Fountain ' s technology enactment framework, the authors integrated inter-organizational factors, such as trust, knowledge sharing, and quality of outsourcing interfaces, in the model and added organizational culture alignment between service providers and public administration to enhance Fountain ' s original framework. Findings – The authors proposed 17 empirically testable propositions to establish the relationships between key variables in IT outsourcing projects in the public sector. Research limitations/implications – The proposed model provides guidance for future research aimed at advancing knowledge of IT outsourcing. Originality/value – The contribution lies in the development of specific variables, such as trust, knowledge, and organizational culture, which are related to building an outsourcing relationship and are used as determinants of the quality of organizational interfaces between public bureaucracies and IT outsourcing providers.

Purpose – The purpose of this paper is to devise recommendations for firms to formulate modes of value capture for their product innovations, ex ante. More specifically, the research question is: how can innovators try to maximize, ex ante, the appropriation of the rent they can derive from their innovating projects? Design/methodology/approach – A theoretical framework is developed and proposed to assess modes of value capture of product innovations and two illustrations are provided to show how the framework can work in practice for innovation projects. Findings – This paper presents a practitioner's view based on the development of an original concept of rent configuration and appropriable rent. Research limitations/implications – In terms of research limitations, the possible endogeneity of intellectual property protection and the timing of were not considered. Practical implications – The framework allows a set of predictions regarding modes of value capture for product innovators. Originality/value – The paper's contribution lies in the proposal of an integrative framework based on the concept of rent configuration, separating analytically three dimensions of innovation value, namely volume, profit and duration. This concept allows the authors to present a richer set of recommendations in comparison to previous frameworks, in order to avoid adopting the form of a yes/no decision tree that tends to over simplify the issues at stake. The authors also contemplate not only erosion effects, but also amplification effects on the rent, which constitutes another contribution of this paper.