François Duhamel

Novel Noncompetitive IL-1 Receptor-Biased Ligand Prevents Infection- and Inflammation-Induced Preterm Birth.

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.

G-Protein–Coupled Receptor 91 and Succinate Are Key Contributors in Neonatal Postcerebral Hypoxia-Ischemia Recovery

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 E2–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 E2–prostaglandin E receptor 4 to govern expression of major angiogenic factors. We propose that pharmacological intervention targeting GPR91 could improve post-HI brain recovery.

p75 Neurotrophin Receptor Participates in the Choroidal Antiangiogenic and Apoptotic Effects of T-Lymphocyte–Derived Microparticles

PURPOSE Choroidal neovascularization (CNV) is a major cause of vision loss in which choroidal vessels penetrate the RPE-an important source of growth factors, including nerve growth factor (NGF), whose activation via the p75NTR receptor promotes apoptosis and inhibits angiogenesis. We demonstrated previously that human T-lymphocyte-derived microparticles (LMPs) significantly inhibit angiogenesis in several models of ocular neovascularization. We investigated how LMPs modulate pro- and antiangiogenic microenvironments during choroidal angiogenesis. METHODS Antiangiogenic effects of LMPs were investigated using a rat model of choroidal angiogenesis. The impact of LMPs on expression of major angiogenic factors was assessed by real-time quantitative PCR (qPCR). To determine whether p75NTR signalling was implicated in LMPs-induced activities, we used a specific antibody and short hairpin RNA (shRNA) targeting p75NTR. Cellular apoptosis was determined via evaluation of activated caspase-3 and annexin V binding. RESULTS The LMPs time-dependently inhibited choroidal angiogenesis by more than 64% after 48 hours of treatment. Removal of the RPE from choroidal explants abolished the antiangiogenic effects of LMPs. The mRNA levels of pigment epithelium-derived factor (PEDF) and NGF were increased significantly following LMPs treatment of intact, but not RPE-removed choroids. Downregulation of PEDF and p75NTR significantly blocked the antiangiogenic effects of LMPs. Finally, induction of choroidal endothelial cell apoptosis by LMPs was dependent on p75NTR. CONCLUSIONS We demonstrate for the first time to our knowledge that LMPs markedly inhibit choroidal angiogenesis via mechanisms that are dependent on the integrity of the RPE, and that are mediated largely by the PEDF and proapoptotic activities of p75NTR.

Restoration of renal function by a novel prostaglandin EP4 receptor-derived peptide in models of acute renal failure

Acute renal failure (ARF) is a serious medical complication characterized by an abrupt and sustained decline in renal function. Despite significant advances in supportive care, there is currently no effective treatment to restore renal function. PGE(2) is a lipid hormone mediator abundantly produced in the kidney, where it acts locally to regulate renal function; several studies suggest that modulating EP(4) receptor activity could improve renal function following kidney injury. An optimized peptidomimetic ligand of EP(4) receptor, THG213.29, was tested for its efficacy to improve renal function (glomerular filtration rate, renal plasma flow, and urine output) and histological changes in a model of ARF induced by either cisplatin or renal artery occlusion in Sprague-Dawley rats. THG213.29 modulated PGE(2)-binding dissociation kinetics, indicative of an allosteric binding mode. Consistently, THG213.29 antagonized EP(4)-mediated relaxation of piglet saphenous vein rings, partially inhibited EP(4)-mediated cAMP production, but did not affect Gα(i) activation or β-arrestin recruitment. In vivo, THG213.29 significantly improved renal function and histological changes in cisplatin- and renal artery occlusion-induced ARF models. THG213.29 increased mRNA expression of heme-oxygenase 1, Bcl2, and FGF-2 in renal cortex; correspondingly, in EP(4)-transfected HEK293 cells, THG213.29 augmented FGF-2 and abrogated EP(4)-dependent overexpression of inflammatory IL-6 and of apoptotic death domain-associated protein and BCL2-associated agonist of cell death. Our results demonstrate that THG213.29 represents a novel class of diuretic agent with noncompetitive allosteric modulator effects on EP(4) receptor, resulting in improved renal function and integrity following acute renal failure.

Lymphocytic Microparticles Modulate Angiogenic Properties of Macrophages in Laser-induced Choroidal Neovascularization

Pathological choroidal neovascularization (CNV) is the common cause of vision loss in patients with age-related macular degeneration (AMD). Macrophages possess potential angiogenic function in CNV. We have demonstrated that human T lymphocyte-derived microparticles (LMPs) exert a potent antiangiogenic effect in several pathological neovascularization models. In this study, we investigated the alteration of proangiogenic properties of macrophages by LMPs treatment in vitro and in vivo models. LMPs regulated the expression of several angiogenesis-related factors in macrophages and consequently stimulated their antiangiogenic effects evidenced by the suppression of the proliferation of human retinal endothelial cells in co-culture experiments. The involvement of CD36 receptor in LMPs uptake by macrophages was demonstrated by in vitro assays and by immunostaining of choroidal flat mounts. In addition, ex vivo experiments showed that CD36 mediates the antiangiogenic effect of LMPs in murine and human choroidal explants. Furthermore, intravitreal injection of LMPs in the mouse model of laser-induced CNV significantly suppressed CNV in CD36 dependent manner. The results of this study suggested an ability of LMPs to alter the gene expression pattern of angiogenesis-related factors in macrophages, which provide important information for a new therapeutic approach for efficiently interfering with both vascular and extravascular components of CNV.

Receptors of intermediates of carbohydrate metabolism, GPR91 and GPR99, mediate axon growth

During the development of the visual system, high levels of energy are expended propelling axons from the retina to the brain. However, the role of intermediates of carbohydrate metabolism in the development of the visual system has been overlooked. Here, we report that the carbohydrate metabolites succinate and α-ketoglutarate (α-KG) and their respective receptor—GPR91 and GPR99—are involved in modulating retinal ganglion cell (RGC) projections toward the thalamus during visual system development. Using ex vivo and in vivo approaches, combined with pharmacological and genetic analyses, we revealed that GPR91 and GPR99 are expressed on axons of developing RGCs and have complementary roles during RGC axon growth in an extracellular signal–regulated kinases 1 and 2 (ERK1/2)-dependent manner. However, they have no effects on axon guidance. These findings suggest an important role for these receptors during the establishment of the visual system and provide a foundational link between carbohydrate metabolism and axon growth.